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3 1293 01037 9968

This is to certify that the

dissertation entitled
Structure and Molecular Regulation of

the Lignin Peroxidase Genes from
the White-Rot Fungus 'I'rametes versicolor

presented by
Andrew James Kavanaugh Black

has been accepted towards fulfillment
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Ph . D. degree in Microbiology

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STRUCTURE AND MOLECULAR REGULATION OF
THE LIGNIN PEROXIDASE GENES FROM
THE WHITE-ROT FUNGUS 'I'RAMETES VERSICOLOR
By

Andrew James Kavanaugh Black

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology

1992

ABSTRACT

STRUCTURE AND MOLECULAR REGULATION OF
THE LIGNIN PEROXIDASE GENES FROM
THE WHITE-ROT FUNGUS TRAMETES VERSICOLOR

By

Andrew James Kavanaugh Black

White-rot fungi are among the few organisms able to degrade all three
constituents of wood: cellulose, hemicellulose and lignin. Lignin peroxidases (LIPS),
produced by white-rot fungi are involved in the key reactions leading to
depolymerization of the lignin polymer. However the LIP gene structure for lignin
peroxidases and their molecular regulation has only been characterized for one of the
white-rot fungi, Phanerochaete chrysosporium. Little is known about the gene structure
or molecular regulation of another well studied white-rot fungus, Trametes versicolor.

Six lignin peroxidase genes (VLGI-VLG6) were isolated from a genomic lambda
library of T. versicolor strain ATCC 12679. DNA sequence analysis of the genomic
sequence for VLGI and VLGZ, as well as cDNA sequences for VLGZ, revealed
substantial stretches of conserved coding sequence between the LIPs of T. versicolor, P.
chrysosporium, and Phlebt'a radiata.

Study of the molecular regulation of the cloned VLG genes induced by growth in
nitrogen limiting media revealed that the major LIP transcript, VLGZ, was induced
coincidentally with LIP enzyme activity. Also LIP activity was completely repressed by
low levels of Mn(II) (2.6 ppm). These results stimulated a study on the molecular

regulation of the VLG genes induced during growth on poplar and pine wood. No such

study has ever been done for any of the white-rot fungi. Study of the molecular
regulation of lignin peroxidase genes in wood revealed that three genes, VLGZ, VLG5,
and VLG3 were induced under various conditions in wood cultures. Differential gene
regulation was found for VLGZ and VLGS transcripts induced by growth on pine wood.
Transcripts for VLG3 were only induced under high nitrogen conditions in poplar wood

grown cultures.

iii

To my mother and father;

and my wife and children.

iv

ACKNOWLEDGMENTS

I would like to thank Dr. C. A. Reddy for his support and guidance, Dr. Thomas
A. Randall for the experimental and intellectual assistance he gave to me, and Dr. Marjan
van der Woude whose stimulating ideas markedly changed the course of my doctoral

research.

TABLE OF CONTENTS

CONTENTS Page
LIST OF TABLES ..................................................................................................... viii
LIST OF FIGURES ..................................................................................................... ix
INTRODUCTION ........................................................................................................ 1
I Wood Structure ...................................................................................... 1
II Microbial Wood Decomposition ............................................................ 5
Bacteria ...................................................................................... 5
Soft-Rot and Brown-Rot Fungi ................................................... 8
White-Rot Fungi ......................................................................... 8
III Basidiomycete Life Cycle .................................................................... 11
IV Li gnin Peroxidases .............................................................................. 12
Discovery ............................................................................................ 12
Activity ............................................................................................... 14
V Manganese Peroxidase ......................................................................... 18
VI Localization of Lignin Degrading Enzymes ......................................... 20
VII Multiple Li gnin Peroxidase Genes ....................................................... 22
VIII Regulation of the Lignin Peroxidase .................................................... 24
Differential Gene Expression .................................................... 28

IX Ligninolytic Enzymes of Trametes
versicolor ............................................................................................. 31
List of References ................................................................................ 35

vi

CHAPTER I Cloning and Characterization of a Lignin Peroxidase Gene From
the White-Rot Fungus Trametes versicolor

Abstract ................................................................................................. 46
Introduction ................................................................................................. 46
Materials and Methods ..................................................................................... 47
Results and Discussion ..................................................................................... 47
Acknowledgements .......................................................................................... 52
References ................................................................................................. 52
CHAPTER 11 Structure and Molecular Regulation of VLGZ, a Lignin

Peroxidase Gene from the White-Rot fungus
Trametes versicolor.

Abstract ................................................................................................. 55
Introduction ................................................................................................. 56
Materials and Methods ..................................................................................... 58
Results ................................................................................................. 61
Discussion ................................................................................................. 64
References ................................................................................................. 66
CHAPTER III Regulation of the Lignin Peroxidase Gene Transcripts in the
White Rot Fungus T rametes versicolor Grown in Wood
Abstract ................................................................................................. 82
Introduction ................................................................................................. 83
Materials and Methods ..................................................................................... 85
Results ................................................................................................. 87
Discussion ................................................................................................. 90
References ................................................................................................. 93

SUMMARY AND CONCLUSIONS

Summary and Conclusions ............................................................................. 109
List of References ........................................................................................... 115
APPENDIX I Determination of the Homology Relationships Within the Lignin

Peroxidase Multigene Family of Trametes versicolor, and
Between the Lignin Peroxidase Subfamilies of Trametes
versicolor and Phanerochaete chrysosporium

Appendix I ............................................................................................... 117

vii

LIST OF TABLES
INTRODUCTION

TABLE 1 Percentage of Lignin, Cellulose, and Hemicellulose lost from paper
birch (Betula papyrtfera) and Eastern white pine (Pinus strobus)

after 12 weeks of degradation by white-rot basidiomycete fungi ............ 10
TABLE 2 Catalytic Properties for Lignin Peroxidase Isozymes of Phanerochaete
chrysosporium ....................................................................................... 30
CHAPTER 11

TABLE 1. Effect of Mn(II) concentration on lignin peroxidase activity of T.
versicolor ............................................................................................... 70

viii

FIGURE 1

FIGURE 2

FIGURE 3

FIGURE 4

FIGURE 5

FIGURE 6

FIGURE 1

FIGURE 2

FIGURE 3

FIGURE 4

LIST OF FIGURES

INTRODUCTION
Expanded section of the secondary cell wall ............................................ 2
Chemical linkages for the lignin polymer of spruce wood ........................ 4
Scheme showing ligninase action on 8-1 and 8-0-4 lignin models ......... l3

Catalytic cycle of lignin peroxidase showing varied reduction states of
the heme. ............................................................................................... 15

Proposed scheme for aerobic and anaerobic cleavage of DMHB by P.
chrysosporium lignin peroxidase ............................................................ 17

Relationship between culture parameters and ligninolytic activity during
7 days of growth by P. chrysosporiwn ................................................... 25

CHAPTER I

Restriction maps of the LIP-encoding genomic clones of T.
versicolor ............................................................................................... 47

Complete nucleotide sequence of T. versicolor LIP gene VLG]
and the amino acid sequence .................................................................. 49

Conservation of amino acid sequences between the LIP proteins of P.
Chrysosporium, Ph. radiata, and VLGI of T. verst'color ......................... 51

Comparison of intron positions and the splice junction sequences in LIP
genes of T. versicolor and P. chrysosporium .......................................... 52

ix

FIGURE 1

FIGURE 2

FIGURE 3

FIGURE 4

FIGURE 5

FIGURE 1

FIGURE 2

FIGURE 3

FIGURE 4

FIGURE 5

FIGURE 6

CHAPTER II

Nitrogen depletion versus lignin peroxidase production during growth
of T. versicolor in nitrogen limiting medium .......................................... 72

VLGZ transcript levels on different days of incubation ........................... 74

Concentration of transcripts of VLGI, VLGZ, VLG3, VLG4, VLGS
and VLG6 in poly(A) RNA from day 9 cultures grown on low nitrogen
medium .................................................................................................. 76

Nucleotide sequence of VLGZ and the deduced amino acid sequence
of the protein encoded by this gene ........................................................ 78

Comparison of intron locations between the T. versicolor genes

VLG] and VLGZ, and the P. chrysosporium genes, LIPZ, LIPS,
and LIP6 ................................................................................................ 80

CHAPTER III

Temporal expression of the transcripts from the LIP gene, VLGZ, of
T. versicolor in poplar wood grown cultures ......................................... 97

Regulation of expression of the transcripts of the LIP gene VLGZ of
T. versicolor in Poplar wood medium versus Pine wood medium ........... 99

Temporal expression of the transcripts of the LIP gene, VLG5, of T.
versicolor in Poplar wood medium, and Pine wood medium ................ 101

Differences in total RNA loaded between samples of poplar wood
grown cultures, and pine wood grown cultures ................................... 103

Comparison of lignin peroxidase gene transcript levels in pine vs.
poplar wood ....................................................................................... 1 105

Regulation of expression of lignin peroxidase genes as determined by
northern blot analyses .......................................................................... 107

FIGURE 1

FIGURE 2

APPENDIX I

Homology Relationships amongst the Lignin Peroxidase genomic
clones of T. versicolor .......................................................................... 120

Homology relationship between the LIP subfamilies of P.
chrysosporiwn and T. versicolor .......................................................... 122

xi

INTRODUCTION

I Wood Structure

Wood is composed of three major constituents: cellulose, hemicellulose, and
lignin. Cellulose and hemicellulose make up 50% and 20%, respectively, of wood
biomass while lignin comprises 20-30% of biomass weight (22, 76). Although cellulose
and hemicellulose are readily metabolized by many soil and forest microbes, lignin is
recalcitrant to degradation. Lignin provides the plant cell wall with a water impermeable
barrier and physical rigidity. And the close association of lignin with the cellulose and
hemicellulose fibrils protects these wood polysaccharides from microbial attack.

The basic structure of a wood cell wall is presented in Figure 1. Lignin is
concentrated most heavily in the middle lamellar region between vessels (28). The lignin
in the middle lamellar region provides a cement between vessel elements and prevents
water permeation between vessels. While heavily concentrated in the middle lamellar
region, lignin is most abundant in the secondary cell walls. In birch wood the fiber,
vessel, and ray cell secondary walls contained over 81% of the tOtal cellular lignin (28).
This lignin surrounds the cellulose fibrils, and is intermixed with the hemicellulose, such
that for efficient conversion of the cellulose to glucose the lignin must be
depolymerized.

Lignin is synthesized in the plant cell by diversion of tyrosine and phenylalanine
to produce the three lignin precursors p-coumaryl alcohol, coniferyl alcohol, and sinapyl
alcohol. These lignin phenylpropanoid subunits are dehydrogenated by a plant

peroxidase (E.Cl.11.1.7, donor; hydrogen peroxide oxidoreductase) to produce

 

 

 

Figure l Expanded section of the secondary cell wall. A. The cellulose fibers (CEL), hemicellulose
(HM), and lignin (LIG) within the secondary cell wall. B. Organization of plant cell walls. Vessel
elements (VL) with their secondary cell walls (81, S2, and $3) and primary cell walls (P) are surrounded
by the middle lamellar region (ML). C. Tracheids (from 22).

 

 

phenoxyradicals. Because the unpaired electron is stabilized by it shell electrons in the
phenylpropanoid precursor, multiple resonance forms are produced (10). Polymerization
of the precursor resonance forms results in an extended, high molecular weight polymer
which has a variety of intermonomer linkages (10). Since the Ga and GB carbons in
the phenylpropanoid sidechains can be either in the R or S form, the lignin polymer is
also non-stereospecific (76). The actual lignin molecule is large, 1 x 105 daltons or
greater, and contains a great variety of the linkage types seen in Figure 2.

Angiosperms (hardwoods) and Gymnosperms (softwoods) share the same basic
ultrastructural characteristics described above for the distribution of their lignin within
the cell wall; however, they differ in the types of plant tissues they have and in their
lignin content and type. Softwood tissues have a simple organization, as pictured in
Figure 1. The wood is basically composed of tracheid cells within the xylem which form
the bulk of the conductive tissue within the wood. Hardwoods are composed of three
basic tissue types within the xylem; rays, vessels, and fibers. These tissue types are
organized within a complex network which differs depending upon the tree genus. In
both hardwoods and softwoods these xylem elements are not only the conductive tissues,
but provide physical rigidity to the tree as well. Gymnosperms generally have a higher
lignin content than angiosperms. For example aspen wood (Populos tremuloides), an
angiosperm, has a lignin content of 22% while Pine wood (Pinus banksiana), a
gymnosperm, has a lignin content of 29.9% (22). Also the phenylpropanoid subunits are
different between softwoods and hardwoods. Gymnosperms contain mostly guaiacyl
lignin, while angiosperms contain widely varying ratios of syringyl and guaiacyl lignin.
Guaiacyl lignin is composed mostly of coniferyl alcohol subunits with smaller amounts
of coumaryl and sinapyl alcohol (ratio for spruce lignin is 80:14:6, respectively) (76).
Syringyl lignin contains relatively high levels of sinapyl and coniferyl alcohol, with
smaller amounts of coumaryl alcohol. The various tissue types of the hardwoods also

contain various concentrations and types of lignin. Fiber cells are the predominant tissue

 

 

 

 

 

 

 

Figure 2 Chemical linkages for the lignin polymer of spruce wood (from 1).

 

fir

 

type in birch xylem. They contain 77% of the xylem's lignin and are 19-22% lignin.
They are also almost 90% syringyl lignin and only 10% guaiacyl lignin. The vessel cells
are more like softwoods in having a higher lignin content, 24-28% lignin, and contain
predominantly guaiacyl lignin. Because of the different lignin types, softwood is harder
to delignify and takes longer to pulp than hardwoods (28).

Possibly related to the relative difficulty of chemically degrading guaiacyl lignin
versus syringyl/guaiacyl lignin, white-rot fungi also generally degrade gymnosperrn
softwoods less effectively than angiospenn hardwoods (40). Trametes versicolor
degrades angiospenn wood (from Eryrt’thrina cristagalli), which contains guaiacyl lignin
levels similar to gymnosperrns, at the same rate as it would a true gymnospenn wood
(40). The faster rate of degradation of syringyl/guaiacyl lignin is due to the faster
depolymerization of syringyl/guaiacyl lignin than guaiacyl lignin by white-rot fungi.
Phanerochaete chrysosport‘um was able to depolymerize both synthetic and natural
syringyl/guaiacyl lignin into smaller dioxane:water—DMF soluble material faster than it
was able to fragment guaiacyl lignin. However once fragmented, the conversion rate of
the guaiacyl and syringyl/guaiacyl lignin monomer and dimer molecules to C02 occurred
at the same rate. The difference in the ability of white-rot fungi to depolymerize
guaiacyl versus syringyl/guaiacyl lignin is most likely due to the differences between the
linkages between the monomers, with guaiacyl lignin having a more condensed structure

with more C-C bonds than C-O-C bonds at C5 (26).

H Microbial Wood Decomposition

Bacteria

Bacteria within the wood decomposition community can be found within several
ecological niches. Bacteria have been categorized into four basic areas. 1) Bacteria that
utilize the cell contents of ray vessel and parenchyma cells without affecring the

structural integrity of the wood fibers and cell walls. 2) Bacteria that degrade the cell

walls of wood directly. 3) Bacteria that are associated with other wood degrading
organisms and have a mutualistic relationship with them. 4) Bacteria that are
antagonistic to resident wood degrading organisms and inhibit their growth (36).

The identification and isolation of bacteria which can attack the wood cell wall
has not been as successful as the identification and isolation of fungal wood degraders.
At this time no single bacterial isolate has been found which when grown in pure culture
can completely mineralize polymeric lignin. Yet bacterial consortia have been described
from wood and soil samples which can partially degrade lignin.

Forney and Reddy (29) identified mixed bacterial consortium, isolated from
tropical soil samples, which could degrade kraft lignin. This bacterial consortium
appeared to mineralize 2-14C-synthetic lignin albeit to a limited extent. Schmidt et. al.
(80) in a study of 57 bacteria isolated from wood, soil, and lake samples found that only
a few strains could colonize the non-lignified wood cell membranes of bordered pits,
parenchyma cells, and epithelial cells. Under laboratory conditions these Actinomycetes,
Bacilli, and Cellulomonas produced only a minimal loss of weight from the lignified
woody substrates.

Tunneling bacteria are perhaps the best examples of bacteria which may be able
to degrade lignin completely. Tunneling bacteria are most likely gliding bacteria of the
Myxobacteriales or Cytophagales groups (17). The bacteria gain access to the cell wall
by localized dissolution of the substrate, and then form tunnels throughout the S3 and 82
layers of the tracheids and vessels. It has not been possible yet to purify these interesting
tunneling bacteria, but in mixed cultures they have been shown to mineralize lignin,
labelled with 14C at either the ring or side chains, to ”cm. A total maximal reduction
of 11% was found for ring labelled lignin and 10.4% for side chain labelled lignin after
16 days cultivation of the bacterium. While it has not been possible to conclusively
prove that bacteria in pure culture mineralize l4C-lignin, bacteria undoubtedly play a

large, if as yet undefined, role in the wood degradation community.

Even if bacteria do not directly mineralize lignin, mutualistic associations
between bacteria and fungi maybe important for lignin degradation. For example
pretreatment of wood with cellulase-negative mutants of the white-rot fungus Phlebia
gigantea was effective in allowing the cellulolytic bacteria Cellvibrio vulgaris,
Sporocytophaga myxococcoides, and S treptomyces olivaceus access to the carbohydrates
within the cell walls. WithOut pretreatment bacterial degradation was much reduced
(41).

In natural wood consortia, bacteria, yeasts, and basidiomycetes can be found
together in mutualistic associations. The role which each of these organisms plays in the
decay community has not yet been completely elucidated. The bacteria may supply
vitamins or other nutrients, and some of the bacterial consortia appear to fix atmospheric
nitrogen (6). This is especially interesting considering the fact that wood contains very
low levels of available nitrogen in comparison to the carbon sources available in the form
of cellulose and hemicellulose. In experiments combining two species of nitrogen-fixing
Enterobacteria, two species of yeasts (Pichia pinus, and Saccharomyces bailit), and the
white-rot fungus Trametes versicolor on mixed western white pine, fir, spruce, and
western red cedar wood chips, the fungal growth rate was increased over 200% as
compared to the growth of T. versicolor alone on the wood chips. Furthermore the extent
of weight loss for the wood inocula increased 1-10%, indicating greater efficiency of
wood degradation by the consortia than by any one species alone. The bacteria and
yeasts were strongly dependent on fungal enzymatic activity since in scanning electron
micrographs the bacteria and yeasts were only found in wood tracheids where fungi were
actively degrading the cell wall. The bacteria and fungi therefore appeared to be utilizing
the carbohydrates released from the wood by the action of the fungal extracellular

enzymes (6).

Soft-Rot and Brown-Rot Fungi

The important fungi which cause soft-rot of water-logged wood belong
predominantly to the class Deuteromycotina (fungi imperfecti) or Ascomycotina. Soft-
rot fungi colonize the cell wall and depolymerize the cellulose and hemicellulose with
concurrent lignin depolymerization (22). Brown-rot fungi belong predominantly to the
class Basidiomycotina and are closely related to the white-rot fungi. Brown—rot wood
degradation causes a substantial loss in the cellulose and hemicellulose content of the
wood with little loss in the lignin content. They appear to have little ability to mineralize
the lignin polymer itself. The main modifications to the lignin polymer are
demethoxylation and hydroxylation of aromatic rings. The brown crumbly residue left
after their colonization is mostly lignin. However, the enzymology of brown—rot or soft-
rot lignin degradation has not been studied extensively and very little is known about the
mechanisms or enzymes they employ for lignin depolymerization (55).

White-Rot Fungi

White-rot fungi, like brown-rot fungi, are predominantly basidiomycetes. They
begin colonizing wood by invading the wood cell lumen, utilizing the cell contents for
growth. The cell wall components are degraded sequentially from the cell lumen starting
with the secondary cell wall. The middle lamellar region is generally the last to be
attacked. A common feature of fungal wood degradation is the presence of an exo-
polysaccharide sheath surrounding the hyphal cell and contacting the plant cell wall. The
sheath has several possible functions. It is a contact point between the fungal cell
membranes and the plant cell wall and therefore provides a microenvironment where
fungal enzymes and plant degradation products can traverse the space between the fungus
and plant cell wall. The sheath may contain the depolymerization enzymes for cellulose,
hemicellulose, and lignin degradation within a microenvironment optimal for their
activity (70). Phanerochaete chrysosporium and Trametes versicolor were the only fungi

out of five white-rot fungi, (the others being Phellinus pini, Xylobolus frustulatus, and

Pycnoporus sanguineus), for which the hyphal polysaccharide sheaths contained cellular
products from autolysis of fungal cells (71). Therefore, at least for these two fungi, lysis
of cells may be an important mechanism of releasing lignin, cellulose, and hemicellulose
degrading enzymes to the plant cell wall.

There are two different strategies which white-rot fungi employ for lignin
removal: 1) simultaneous degradation of all three wood components; 2) preferential
attack on the lignin and hemicellulose either before, or to the exclusion of cellulose
degradation. White-rot fungi which simultaneously decay all three major polymeric
components of wood exhibit several distinctive characteristics. Holes and erosion
troughs in the cell walls of the wood are evident near the fungal hyphae indicating
degradation of all wood components at once in one localized area. Degradation of the
wood proceeds progressively from the cell lumen outward to the secondary cell wall, and
the middle lamellar region is attacked last. The tracheids in gymnospenns, and
parenchyma, vessel, and ray cells in angiosperms stay intact until the final stages of
decay. In general, as shown in Table 1, the percentage of loss for lignin, cellulose (as
represented by glucose), and hemicellulose (as represented by losses in both xylose and
mannose) is approximately equal. Both T. versicolor and P. chrysosporium are
simultaneous lignin degraders (15, 22), although P. chrysosporium utilizes xylose
residues preferentially over glucose and mannose residues.

For fungi which exhibit preferential decay of lignin, no holes or erosion troughs
can be found within cells which have hyphae present in the lumen. Lignin is still
removed progressively from the lumen outward towards the secondary cell wall, and
finally towards the middle lamellar region; but the cell wall stays intact. Scanning
electron micrographs of wood degraded by preferential lignin degraders shows the lignin

has been removed from the wood, while the cell structure is unaffected. The progressive
9 delignification of the middle lamellae causes softwood tracheids, and hardwood vessels

and rays to separate from each other (4). A distinctive feature of this type of decay is

10

Table 1 Percentage of Lignin, Cellulose, and Hemicellulose lost from paper birch
(Betula papyrifera) and Eastern white pine (Pinus strobus) after 12 weeks of degradation
by white-rot basidiomycete fungi.

 

 

Fungus Wood Percentage Loss8
of
A. Simultaneous Degraders
Li gnin Glucose Xylose Mannose
Trametes versicolor Birch 64.6 65.4 68.8 71.7
Pine 35.4 22.1 46.7 1 1.6
P. chrysosporium Birch 72.9 15.1 55.1 0.0
(BKM-F- 1767) Pine 30.5 3.9 44.1 0.0
B. Preferential Degraders
Dichomitus squalens Birch 71.2 43.8 43.5 40.4
Pine 53.0 48.3 56.9 64.9
Heterobasidion Birch 54.6 4.1 5.6 2.2
annosum Pine 24.0 12.6 14.7 19.6
Phellinus pim' Birch 53.9 5.2 12.9 1 1.4
Pine 43.5 10.7 34.5 42.5
Phlebia tremellosa Birch 75.2 4.1 39.4 28.5
Pine 38.6 20.2 61.2 41.2
Poria medulla-pants Birch 73.1 0.1 31.9 1 1.5
Pine 38.5 28.6 62.9 35.3

 

 

a Glucose and Xylose/Mannose loss, respectively, represents degradation of the wood
polysaccharides cellulose, and hemicellulose (from 22).

11

that the resulting delignified wood has been depleted of almost all the lignin and
hemicellulose but has not lost a corresponding level of cellulose (5). This is represented
by the high percentage of glucose left in the wood decayed (degraded) by Heterobasidion
annosum, Phlebia tremullosa, and Phellinus pint“ (Table 1). The concomitant loss of
lignin and hemicellulose during preferential lignin degradation is not too surprising
considering the close physical and chemical association of the two polymers within the
cell wall, and the fact that lignin degradation has been shown to require a readily
hydrolyzable carbon source.

111 Basidiomycete Life Cycle

The basidiomycete life cycle is an important factor affecting the interactions
among fungi within the decay community. Basidiomycetes have an unusual
haplodikaryotic vegetative state where the fungal cell contains two (or more) independent
haploid nuclei. These haploid nuclei are derived from two monokaryotic sexually
compatible parental fungi. After plasmogamy (when the fungal hyphae fuse), the parental
nuclei remain separate. Basidiomycetes, unlike Ascomycetes, can and do exist in this
heterokaryotic vegetative state for extended periods of time. Only when conditions for
sporulation are right do the nuclei fuse (karyogamy) within the sexual cells of the
basidiocarp. These diploid basidial cells go through meiosis and daughter haploid nuclei
are deposited within basidiospores which are released to form new monokaryotic
individuals (75).

Two parental hyphae can only complete plasmogamy if they are of the correct
mating type. For heterothallic fungi the parental hyphae must be of different mating
types. Homothallic fungi can self-mate and produce genetically identical basidiospores.
Mating type in the heterothallic fungi is determined by either one (bipolar) or two

(tetrapolar) genes. Thus for a heterothallic, tetrapolar fungus like Trametes versicolor,
9 the monokaryotic parental hyphae must have different alleles for the two mating type

genes (referred to as AB x ab). The dikaryotic hyphae resulting will be genetically and

12

physiologically identical to a diploid cell with the mating type alleles AaBb. For T.
versicolor the number of alleles for each of the two mating type genes is quite large so
that the chance encounter of two identical mating types is rare (91). Dikaryotic
heterokaryons are antagonistic to every other genetically non-identical mono or dikaryon
of the same species. If two such non-identical hyphae should meet, plasmogamy will not
result, and a characteristic incompatibility reaction occurs.

IV Lignin Peroxidases

Discovery

The search for peroxidases involved in lignin degradation began with the finding
that hydrogen peroxide was required in the extracellular environment for lignin
degradation to occur (31). The production of H202 by the white-rot fungus P.
chrysosporium was shown to coincide with the cessation of primary growth and the
concomitant degradation of synthetic 14C labelled coniferyl alcohol lignin. It was
further shown that the hydroxyl radical scavenging agents mannitol, benzoate, and BHT
markedly inhibited lignin degradation. This report was substantiated by Kutsuki and
Gold (59) who showed that catalase inhibited lignin degradation as well as the hydroxyl
radical scavengers, thereby strengthening the relationship between hydrogen peroxide
production and lignin degradation.

These results led to the discovery of an enzyme, ligninase, later called lignin
peroxidase (abbreviated as LIP), that could degrade synthetic and natural lignins and
required for activity (34, 85). Isolation of this lignin peroxidase showed it to be a
42,000 dalton heme protein with one protoheme IX per molecule and an isoelectric point
of 3.5. The enzyme was found to catalyze the oxidation of veratryl alcohol (3,4
dimethoxybenzyl alcohol) to veratraldehyde at a Vrnax of 84 pmol veratryl alcohol
oxidized per min per mg of protein at a pH of 3.5. Maximal enzyme activity was found
'with 0.15 mM H202 and the Km for H202 was 30 pM. The LIP enzyme was also

found to catalyze the cleavage of B-1 and 15—0-4 lignin model compounds (see Figure 3)

13

 

 

 

Figure 3 Scheme showing lignin peroxidase action on A. fl-l and B. B-O-4 lignin models (from 86).

 

 

14

with the oxygen molecule coming from molecular oxygen and not hydrogen peroxide
(60, 86). Most of the spectral and kinetic studies have been performed on this particular
isozyme (designated H8) which was the first lignin peroxidase to be isolated, and the
catalytic mechanism for this enzyme has been elucidated. These extensive studies are
summarized below from several excellent reviews which have already been published
(10, 55, 84).

Activity

Assays for LIP activity have utilized the oxidation of veratryl alcohol to
veratraldehyde since the lignin peroxidase enzyme cannot oxidize veratraldehyde further.
The stoichiometry is one mole of H202 consumed per mole of veratraldehyde produced.
Saturation kinetic studies using both veratryl alcohol and H202 indicate that the reaction
mechanism is ping-pong. Spectral changes of lignin peroxidase observed upon reaction
with H202, but not with veratryl alcohol, suggest that lignin peroxidase first reacts with
H202 to produce an oxidized enzyme intermediate compound I (Figure 4). Compound I
with H202, but not with veratryl alcohol, suggest that lignin peroxidase first reacts with
then performs a single electron oxidation of aromatic structures such as those found in
lignin, producing a cation radical in the carbon center of the lignin polymer and the
oxidized compound II.

LIP has been found to catalyze the depolymerization of dimeric lignin model
compounds by one electron oxidations of the aromatic carbon centers. In studies on
lignin model compounds, single electron oxidations of the aromatic carbon centers
produces unstable cation radicals which depolymerize into various benzylic compounds.
(39, 52, 81). The cation radicals formed in the methoxylated benzene rings lead to
cleavage of the propyl carbon bridge linking the two aromatic rings at the Cor-CB
position (Figure 5). The presence of alkoxylated groups on the aromatic ring is
necessary for the oxidation to occur since benzene rings with fewer than two methoxyl

groups are not oxidized by LIP.

 

 

RH 1-le

' 9 9
C56“ CR“
CompoundWOmpound I

R- RH

Figure 4 Catalytic cycle of lignin peroxidase showing varied reduction states of the heme (from 84)‘.

 

 

16

 

 

3‘0
‘3 Mom 00“:

QVIN,
OCH,
0’z +H’
/

I I
[202+ ’2 ”2°? 0“

Figure 5 Proposed scheme for aerobic and anaerobic cleavage of DMHB by P. chrysosporium lignin
peroxidase (from 39).

 

 

17

The alkoxyl groups (or hydroxyl groups) stabilize the positive charge on the
aromatic ring and therefore promote formation of aromatic cation radicals (39). The
aryl-aldehydes formed result from whichever carbon (either Ca or CB) accepts the free
electron. Since the aromatic cation radicals can also act as electron transfer agents, the
action of LIP on one end of the molecule may spread to aromatic centers at distal ends of
the lignin polymer. The result would be depolymerization of lignin in regions of the
molecule which are inaccessible to the 42 kd LIP. Ultimately the hydroxylated radicals
must be reduced further by one electron to prevent their repolymerization.

Although a mechanism for lignin depolymerization by UPS has been elucidated it
has also become clear that for lignin depolymerization to occur other enzymes must be
active. Haemmerli et al., (37) showed that alkali straw lignin, HCl-dioxane extracted
straw lignin, and milled wood lignin were oxidized by UPS of P. chlysosporium and
added H202. The result was not depolymerization, but further polymerization.
Sarkanen et al., (78) extended this result by showing LIP, in the presence of H202, was
able to polymerize the monomeric lignin precursor coniferyl alcohol into an insoluble
high molecular weight synthetic lignin. LIP produced aromatic cation radicals from the
coniferyl alcohol, and these were able to form a dehydrOpolymerizate. These
observations are not surprising given that there was a high concentration of substrate
(coniferyl alcohol) and the product (dehydropolymerizate) was insoluble and therefore
removed from solution by precipitation. LIPS are able to cause net depolymerization of
synthetic lignin if three conditions are met: (1) The synthetic lignin is in a dilute
suspension in HZO/dimethylformamide in order to increase its solubility, (2) the H202
concentration is maintained at a low level to assure that aromatic free radical
concentrations are kept low, and (3) veratryl alcohol is added to assist in stabilizing the

LIP and extending its active life (38).
A The importance of LIP to lignin degradation has been further substantiated by

studies on a LIP negative mutant (8, 9). This mutant (Ii/75) has no detectable LIP

18

activity, yet retains the important ligninolytic enzymes glucose oxidase and manganese
peroxidase (see section VI and VII below). The loss of LIP activity was correlated with
a substantial decrease in mineralization of l4C-labeled synthetic lignin (6%), as
compared to 31% degradation observed with the wild type.

The equilibrium between the forward and reverse reactions catalyzed by LIP may
be made to favor the forward direction by the removal of aromatic lignin degradation
intermediates. The rapid removal of substituted quinones by quinone reductases, could
possibly fulfill this role. Both extracellular and intracellular quinone reductases have
been identified, and an intracellular NADH:quinone oxidoreductase has now been
purified from P. Chrysosporium (14). Further studies on these enzymes and the role they
play in lignin depolymerization are being investigated.

V Manganese Peroxidases

A second major class of peroxidases involved in lignin degradation were
discovered soon after the initial isolation of LIP. They contain one molecule of heme as
iron protoporphyrin IX per enzyme molecule. The activity of the enzyme on lignin
model compounds was found to be dependent on both H202 and Mn(II), and its
principal enzyme activity appeared to be the oxidation of Mn(II) to Mn(III) (41). At a
pH Optimum of 5.0 the enzyme oxidized Mn(II) to Mn(III) at a higher rate than any other
available substrate, and the Mn(III) complexed with lactate, or other a-hydroxy acids,
was an obligatory intermediate in the oxidation of polymeric dyes or lignin model
compounds by the enzyme. The enzyme has been called manganese peroxidase (MNP)
or manganese-dependent LIP (32) and have a slightly higher M, of 46,000 daltons than
LIP. The catalytic cycle of MNP is similar to LIP in that the intermediate compound
MNP I is formed by a two electron oxidation of the resting state enzyme by H202. The
subsequent individual one electron oxidations of two Mn(II) molecules produces two

Mn(III) molecules and the native state MNP enzyme (88, 90).

19

Considering that both LIP and MNP utilize H202 to bring about one electron
oxidations and depolymerization of the lignin polymer, the discovery that Mn(II) levels
regulate both LIP and MNP enzyme levels in P. chrysosporium is highly significant
(127). In Mn(II) free cultures LIP activity appeared earlier, and was 2.5 times higher
than that in basal media containing 11.15 ppm Mn(II), while MNP activity was very low
or undetectable in media containing no Mn(II) and was 24 times higher in media with
11.15 ppm Mn(II). While concentrations below 1.6 ppm stimulated LIP activity,
concentrations above 8.0 ppm suppressed LIP activity. At 40 ppm Mn(II), LIP activity
was completely suppressed. MNP activity was completely suppressed in the absence of
Mn(II) and increased steadily to a peak level at 40 ppm Mn(II). Similar results were
obtained with the white-rot fungi Phanerochaete flavido alba, Phanerochaete magnolia,
Phlebia radiata, Phlebia tremellosa, Phlebia subserialt's, Lentinus edodes, and Phellinus
pini. Yet, even though MNP's and UPS are theoretically able to depolymerize lignin to
the same extent, ligninolytic activity (as measured by mineralization of synthetic lignin)
was more than seven fold higher at low Mn(II) levels (0.32 ppm) than at high Mn(II)
levels (39.88 ppm). Therefore, low Mn(II) levels correlated with low MNP activity, high
LIP activity, and high levels of lignin degradation. High Mn(II) levels correlated with
high MNP activity, low LIP activity, and low levels of lignin degradation. Natural
concentrations of manganese in wood are reported to range from 5 to 200 ppm, (4, 7,
Blanchette; personal communication), however the concentrations of Mn(II) available to
the fungus are not known since Mn(II) can be bound by the wood in an insoluble form.
Still, Mn(II) may play a role in regulating LIP activity in wood. MNP's would act by
oxidizing the available Mn(II) to Mn(III). Mn(III) could then diffuse into the wood and
cause changes in the lignin polymer which would make the lignin more susceptible to
LIP activity. After some time Mn(II) may become depleted around the fungal hyphae,

and the LIPS would be derepressed (7). It is known that white-rot fungi can mobilize the

20

manganese in wood and concentrate manganese in white-rot degraded wood into deposits
with 100 fold greater manganese concentrations than in sound wood (4).

Two different MNP cDNA clones have been isolated from P. chrysosporium;
MP-I, coding for MNP2 (72), and MNP-1 coding for a different as yet unidentified MNP
peak (73). The homology of MP-I to the first LIP enzyme isolated (H8) is 58% at the
nucleotide level, and 65% at the protein level. Sequencing of the genomic clone for
MNP-I indicates that while Splice junction sequences are the same, the position of the
introns between UPS and MNP's have not been conserved (35). It has been found that
Mn(II) levels regulate the transcription of the MNP-I gene (12). Very low levels of
MNP-I mRNA were present at 0 ppm Mn levels, but as Mn(II) concentrations were
increased, the level of MNP-I transcript also increased substantially to a peak at 180 pM
MnSO4, (10 ppm Mn(II)), with transcripts appearing within 40 mins of the Mn(II)
addition (12).

VI Localization of Lignin Degrading Enzymes.

Decomposition of 14C-lignin to 14C02 by P. chrysosporium occurs
simultaneously with the depolymerization of lignin to smaller molecular weight
substances (13). The progressive changes in the molecular size of lignin indicated that
the depolymerization was not a separate temporal event during lignin degradation, and
that smaller molecular weight lignin degradation products are catabolized concomitantly
with lignin depolymerization.

Part of this system requires the production of hydrogen peroxide for its activity.
The search for the enzymatic source of the H202 production has been an important goal
in elucidating the complete lignin degradation system. Production of H202 by P.
chrysosporium has been shown to occur within the periplasmic space of the ligninolytic
cells (30). The H202 produced there is thought to diffuse out to the extracellular
environment for lignin degradation, thus protecting the intracellular cytoplasm from

harmful oxidations by the reactive H202. Glucose oxidase (GOX), an intracellular

21

H202 producing enzyme was discovered by Kelley and Reddy (50). GOX produces
H202 from D-glucose, or less efficiently, from L-sorbose, D—cellobiose, D-maltose, D-
mannose, and D-xylose. All of the GOX activity was associated with the cellular
fractions from ligninolytic cultures of P. chrysosporium and, therefore, no glucose-1-
oxidase activity could be detected in the extracellular culture fluid. Since H202
production does occur in the periplasmic space of cells, the cellular location of GOX
activity does not preclude its activity in lignin degradation. And its use of glucose, the
main metabolite from cellulose degradation, further supports its role in lignin
degradation.

Still, the search for an extracellular H202 producing enzyme continued. One was
found by Kersten and Kirk (51) which utilized glyoxal or methylglyoxal to produce
H202 in ligninolytic cultures of P chrysosporium. Significantly the substrates glyoxal
and methylglyoxal are also present in the extracellular environment of ligninolytic
cultures. However, the enzyme glyoxal oxidase (GLOX) is a minor protein component
of the extracellular fluid of ligninolytic cultures, and, therefore, whether it can supply all
of the required H202 for ligninolytic activity is debatable.

Many studies have attempted to determine the location of LIP activity during
lignin degradation. Various groups have identified LIP as being located predominantly
within the cell, in the periplasmic space, or within the wood. The first studies using
immunogold-labelled antibodies to LIP found it located predominantly in either the
intracellular vesicles, or bound to the plasma membrane in the periplasmic space (83).
These results corroborated earlier studies which reported a relationship between the
strong binding of lignin to the fungal mycelium and lignin degradation (l3).
Improvements in staining and fixing techniques, however, showed that while
depolymerized lignin may attach to the mycelial membrane, contact between fungal
hyphae and degradation of the plant cell wall are not necessary (15). No differences are

noted between degradation patterns of hyphae adpressed to the cell wall, and those

22

located in the center of the lumen. Degradation produces characteristic erosion troughs
and decay zones where the attack on wood polymers is localized within a small region
and progresses outward. Subsequent studies have found that LIP is located not only
within the fungal periplasmic space, fungal slime layer, and membranous materials
bound to wood cell walls, but also is found to have penetrated into decay zones within
the wood.

The decay zones are thought to be caused by fungal depolymerization of the 82
layer. LIP enzyme was found to consistently penetrate the 82 layer within the decay
zone and concentrate at the interface between degraded and undegraded wood. Also LIP
was found to accumulate within erosion troughs. The enzyme was never found to have
penetrated undecayed wood. Therefore the partial depolymerization of wood in the S3,
82, or 81 cell wall layers allows the LIP (Mr 42,000) to penetrate the wood and
concentrate in regions where active lignin depolymerization is taking place (15).
Parallel studies using immunogold labelled antibodies to MNP showed a similar
distribution to MNP localization. The polyclonal antibodies made against MNPs did not
cross-react with LIP. Double labelling experiments using 5 nm gold probes for MNP and
15 nm gold probes for LIP showed that both peroxidases were localized within decay
zones and concentrated at interfaces between degraded and undegraded wood (16).
These results support a role for MNP in lignin degradation as has been claimed by other
investigators (32).

VII Multiple LIP Genes

Within three years of the initial discovery of LIP, it was found that P.
chrysosporiwn produces multiple LIPS. Under carbon sufficiency, nitrogen limitation
conditions (56 mM glucose, 2.2 mM NH4), and dimethyl succinate buffer P.
chrysosporium strain BKM-F 1767 produces ten extracellular hemeproteins, six of which
ishow LIP activity (54). The hemeproteins were separated by high pressure liquid
chromatography (HPLC) and were arbitrarily named H1 to H10 according to their

23

elution from the column. LIP isozymes H2, H8, and H10 were predominant under these
conditions. LIP H8, induced to the highest level under these conditions, corresponds to
the LIP isozyme first isolated and was the enzyme used for all the preceding kinetic and
mechanistic studies. The other two hemeproteins, H2 and H10, were shown to be
homologous to H8 by their cross reactivity on western blots to polyclonal antibody
prepared against H8. Under similar nitrogen limitation conditions utilizing acetate buffer
instead of dimethyl succinate buffer LIP isozyme H6 predominates. Other workers have
reported up to 15 separate UPS and six MN Ps isolated from nitrogen and carbon starved
cultures of P. chrysosporium BKM-F 1767 (62).

Rapidly following the discovery of the first LIP enzyme, H8 (85), the first LIP
cDNA clones were isolated (93). It was originally assumed that the multiple LIP
isozymes arose by glycosylation and other post-translational modifications of a single
LIP gene product. But sequencing of the LIP clones (LIP's) showed that each of the
hemeprotein LIPs may have a distinct gene coding for it. In order to clarify the
relationships between the various LIP enzymes, and their corresponding genes,
Boominathan and Reddy (10) have proposed the following nomenclature. LIP enzymes
should be designated LIPl-LIP6 instead of the arbitrary heme peak assignments Hl-HIO.
The corresponding enzymes would then be LIPl (H1), LIP2 (H2), LIP3 (H6), LIP4
(H7), LIPS (H8), and LIP6 (H10). The genes encoding these enzymes are given the
same name but always in italics (LIPI-LIPO). Sequence analysis of the cDNA clones of
LIP2 and LIP6, previously designated, respectively, CLG4 and CLGS (19, 20), and LIPS
(87), showed the genes encoded homologous proteins of mature Mr 37,000 which upon
glycosylation increased to 38,000-42,000. The mature proteins are preceded by signal
sequences of 27 (for LIP2) to 28 (for LIPS and LIP6) amino acids. All the UP genes
p sequenced so far have two conserved regions which correspond to putative proximal and

distal histidine peroxidase active sites.

24

The isolation of genomic LIP clones has defined the relationships between the
UP genes of P. Chrysosporium. Most of the genomic clones isolated are identical to, or
differ only by a few nucleotides from, the LIPS cDNA clone (3, 11, 43, 79, 82, 89).
Studies of the gene structure for the other two major LIP genes, LIP2 and LIP6, have
revealed a subfamily organization between the genes. LIP2 is less closely related to all
the other LIP genes studied so far, with 73% and 72% amino acid homology to LIPS and
LIP6, respectively, as compared to 80% amino acid homology between LIPS and LIP6
(68, 92). Furthermore the position of the introns is more similar between LIPS and LIP6
than either is to LIP2. The close relationship between LIPS and LIP6 is further
substantiated by similar peptidase digestion patterns and close immunological cross
reactivity (27, 62).

The discovery of structurally related LIP genes within a multigene family spurred
the search for LIP gene linkage patterns within the chromosome of P. chrysosporium.
Genetic studies on P. chrysosporium strain ME446 found that this strain contains two
sets of LIP genes clustered within its genome (74). The clusters were defined by an
RFLP-based genetic map which showed that at least some of the UP genes occur on the
same chromosome. To date, only two LIP genes of P. chrysosporium strain ME446 have
been shown to be closely linked (separated by 1.3 kb of intergenic DNA) (43). The
genes are orientated for convergent transcription and are highly homologous with amino
acid similarities of 91.7%.

VIII Regulation of the LIP Enzymes and Genes

The ligninolytic enzyme system of P. chrysosporium has long been recognized to
be induced by nitrogen starvation. The cessation of growth by the fungus caused by
nitrogen starvation coincides with the appearance of ligninolytic enzymes which can
degrade 14C labelled synthetic lignins (see Figure 6) (53). Within 48 hours of growth,
P. chrysosporium had depleted 90% of the available NH4+ initially present at 2.2 mM,

while glucose had decreased only 60% from an initial level of 56 mM. The nitrogen

25

 

 

 

a
q
.1
.1
.1
d
at

 

 

 

 

 

 

”I grea- .
i
0" ar- 50!’ gm ‘
3
fi
\ Q. d
.. Eam- cot- €00 '4
g e s ‘-
\ § 5 E
R k a 3
“r— 26'- 8”" 0:“ q
)- U
a Q 3 V
‘ d
a S 9
a 4 _ t” d
s‘l' .10” m g
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S E -
2» ‘00.:- Dr- ‘20 a
a
t‘.
2 t
0- 0 0L- 0

 

C“. It“ a (ml

Figure 6 Relationship between culture parameters and ligninolytic activity during 7 days of growth by
P. chrysosporium (from 53).

 

 

26

limitation signal was sufficient to induce ligninolytic activity, and did not require lignin
to be present. Similar results were also obtained with T. versicolor indicating the broad
relevance of this induction signal to white-rot fungi. Based upon the ubiquitous nature of
the induction of ligninolysis by nitrogen starvation, and the fact that wood is a nitrogen-
poor growth substrate, it is generally assumed that nitrogen starvation is an
environmental signal for the presence of wood as a growth substrate and the need to
express the fungal ligninolytic enzymes (53).

It has also been found that high levels of initial NH4+ causes a substantial
decrease in overall lignin decomposition. Raising the initial NH4+ concentration from
2.4 mM to 24 mM causes a 65-75% decrease in the ability to mineralize 14C-labelled
synthetic lignin. Incubation of the fungus under these high nitrogen conditions causes
glucose starvation before the available nitrogen has become depleted (56). However
carbon limitation is also a signal for induction of ligninolytic activity. With initial levels
of 4.4 mM cellobiose and 7.7 mM nitrogen, carbohydrate becomes limiting first. Under
these conditions ligninolytic activity appears even earlier on day 2 than the appearance of
ligninolytic activity on day 3 when limiting nitrogen (2.6 mM) is used (44).

While P. chrysosporium shows almost complete repression of the lignin
degradation system by nitrogen and carbon sufficiency (34 mM glutamate, 139 mM
glucose), T. versicolor is only moderately repressed in its ligninolytic activity by the
same nitrogen and carbon sufficient medium which had synthetic lignin added to it at the
time of inoculation. And Lentinus edodes and Pleurotus ostreatus are actually stimulated
in their ligninolytic activity by these conditions (61). Therefore, while nitrogen and/or
carbon limitation may be a common induction signal for expression of ligninolytic
activity by white-rot fungi, fungi may differ in their sensitivity to high nitrogen or carbon
‘ levels.

The induction conditions for the LIPS of P. chrysosporium are identical to those

for the entire lignin degradation system. LlPs appear in the extracellular culture fluid

27

coincidentally with the degradation of 14C—labelled lignin; within 2-3 days in nitrogen
limited cultures (2.2 mM nitrogen, 1% glucose), and in 3 days for carbon limited cultures
(6.6 mM nitrogen, 0.15% glucose). In the nitrogen limited cultures, LIP activity could
be further increased two- to five-fold by the addition of lignin or lignin model
compounds, such as the dimeric lignin model compound arylglycerol-B—aryl (13-0-4).
Birch lignin also stimulated the production of H202 which is required for LIP activity
(24). A lignin related compound, veratryl alcohol, was found to be the best inducer of
LIP activity in nutrient-limited cultures. Interestingly veratryl alcohol is synthesized by
white-rot fungi themselves after they shift from primary to secondary metabolism.
Veratryl alcohol is an actual inducer of LIP enzyme expression, and does not simply
protect the LIP enzyme already produced from inactivation. Veratryl alcohol moreover
induces the protein expression of some of the LIP enzymes better than others, since LIP2
was induced 1.8-fold while LIPS was only induced 1.1-fold. However, the overall
induction of LIP activity was still under the control of nutrient limitation since veratryl
alcohol, or lignin, added without nutrient limitation caused no lignin degradation or LIP
induction, and LIP induction could be repressed by the addition of macronutrients even
in the presence of veratryl alcohol (25).

The genetic regulation of LIP enzyme has begun to be studied with both mutants
of P. chrysosporium and by using molecular techniques. Nitrogen-deregulated (der)
mutants of P. chrysosporium which produce high levels of LIP enzymes and degrade
l4C synthetic lignin to 1‘l'COz under conditions of nitrogen sufficiency (24 mM
nitrogen, 1% glucose) have been produced by gamma irradiation (9). Removal of
nitrogen regulation not only allows expression of LIP, MNP, and GOX activity in high
nitrogen media, but the levels of LIP activity in low nitrogen media, (2.4 mM nitrogen,
1% glucose), are increased 4-fold over levels in the wild type parental strain ME446
under identical limiting nitrogen conditions. Since LIP, MNP, and GOX enzymes were

all deregulated in this mutant by gamma irradiation, it suggests a single common

28

regulatory element which controls nitrogen regulation of ligninolytic activity for these
separate families of secondary metabolic enzymes. However the onset of LIP, MNP, and
GOX activity was still delayed in the der mutants until growth had stopped and
secondary metabolism begun (8). Hence, there must be at least two sets of signals for
LIP and MNP gene induction, one for nitrogen repression, and another for the shift to
secondary metabolism. A mutant of P. clu'ysosporium, PSBL-l, has been described
recently (69) which produces 4- to 10 fold higher levels of LIP, MNP, and GIDX
enzymes under not only nitrogen sufficiency, but during primary metabolism (while the
fungus is still growing). Therefore, the genetic signals controlling the shift to secondary
metabolism can be overcome as well.

Differential Gene Expression

Once the existence of multiple LIP genes in P. chrysosporium was realized, the
question arose as to why there were multiple genes for enzymes which seemed to
catalyze the same activity. Studies were begun to determine if there were any regulatory
or enzymatic differences between the many genes. The first indication of differential
gene regulation came from a report by Holzbaur and Tien (42) that while LIP2 specific
transcripts were present in the poly(A)-RNA isolated from both nitrogen- and carbon-
limited cultures, UPS transcripts could only be found in poly(A)-RNA from nitrogen-
limited cultures. The enzyme expression pattern corroborated this result in that nitrogen-
limited cultures had major LIP peaks for LIP2, LIPS, and LIP6. But in carbon-limited
cultures only a single LIP peak, that for isozyme LIP2, could be seen. There are
different hypotheses to explain these results. One hypothesis is that the multiple genes
exist because each gene has a promoter which is differentially activated by a separate set
of environmental signals. This appears to be important because of the diverse
environmental signals the fungus must respond to, and because each environmental
signal apparently activates a separate regulatory system each of which induces a different

subset of UP genes. Under this theory, while different'LlP genes may be induced

29

transcriptionally by different environmental signals, the isozymes coded for by these
genes would all perform the same basic catalytic function.

The other hypothesis to explain the observation of differential gene regulation is
that the different LIP isozymes have some catalytic or substrate binding differences, and
they are differentially regulated so that the enzyme is only made when its specific
catalytic or substrate binding function is required. Evidence in support of this has come
from two studies. The catalytic properties of the LIP enzymes induced under nitrogen
limitation were studied by Farrell et al., (27). Their results are summarized in Table 2.
Surprisingly some of the isozymes have different ranking orders with respect to Km
when tested against veratryl alcohol and the lignin model dimer. For example LIP4 has a
high Km for veratryl alcohol, but only a moderate Km for Model 1. However LIP6 has a
moderate Km for veratryl alcohol but a high Km for Model 1. These differences suggest
that different isozymes have different substrate affinities or different substrate
accessibilities to the active sites. These results were expanded by Dass and Reddy (18)
and correlated with differential expression of LIP enzymes induced when the buffer
dimethylsuccinate (DMS) in nitrogen limited cultures is switched to sodium acetate.
Switching the buffer from DMS to sodium acetate in shaken cultures causes major
changes in the isozyme expression pattern. In shaken DMS cultures, as stated
earlier,LIP2, LIPS, and LIP6 are the major LIP isozymes expressed. In shaken sodium
acetate cultures LIP2 and LIP3 are the major LIP isozymes expressed, and the MNP
isozymes MNP2 and MNP3 become the major hemeproteins expressed in the culture.

Also a new LIP protein, HA, appears as a minor peak when the buffers are
changed. What the buffers DMS and sodium acetate may be signaling to P.
chrysosporium about the environment is Still a mystery, yet the large change in isozyme
(expression indicates that a major change has occurred in gene regulation. The LIP
isozymes were also shown to exhibit a great deal of difference in their enzyme activity

towards different dyes. These polymeric dyes are considered colorimetric indicators of

30

Table 2 Catalytic Properties of Different Li gnin Peroxidase Isozymes of Phanerochaete
chrysosporium

 

 

 

Isozyme Veratryl 1,4-dimethoxy
H202 alcohol Model 1 Benzenec

Kma Km TNb Km TN Km TN
LIPl 13 86 3.20 99 1.43 42 1.50
LIP2 42 250 8.33 78 3.07 132 3.01
LIP3 34 122 6.58 27 1.06 78 4.17
LIP4 77 483 5.46 88 0.76 20 --
LIPS 17 89 1.32 111 0.50 73 8.55
LIP6 24 190 2.78 444 0.43 192 0.84

 

 

Table from Farrell et al., (27)
a Km units are micromolar
b TN = Turnover number (per second)
c Model 1 is a diaryl propane lignin model dimer, [1-(3.4-
dimethoxyphenyl)-2-(o-methoxyphenoxy)-propane—l ,3-diol

31

lignin degradation activity (33) and have been used to isolate lip mutants of P.
Chrysosporium (8, 9). Dass and Reddy (18) used these results, along with those of Farrell
et al., (27), to postulate that the different LIP isozymes play different roles in lignin

degradation.

IX Ligninolytic Enzymes of Trametes versicolor

Trametes versicolor has long been studied for its ability to degrade lignin, and the
volume of research done on its ligninolytic enzyme system is second only to that of P.
chrysosporium. T. versicolor (Coriolus versicolor) is known to degrade the lignin
component of wood completely to C02 (77). The induction conditions for ligninolytic
activity were similar, but not identical to, conditions reported for P. chrysosporium. In
experiments on mineralization of synthetic 14C—lignin overlaid on cellulose plates, T.
versicolor converted over 75% of the lignin to C02 by day 14 when low nitrogen
conditions were employed (2.2 mM NH4). When nitrogen levels were raised to 5.5 mM
NI-I4, the ligninolytic activity was decreased but not repressed completely as seen in P.
chrysosporium. T. versicolor converted 45% of the original lignin to C02 by day 14. A
lag period of four days was observed before ligninolytic activity occurred and the
presence of cellulose was absolutely required for ligninolysis. In the absence of cellulose
no lignin was mineralized (23).

Similar results were also obtained with 14C labelled wheat lignin. The onset of
14C evolution was delayed for 3 days while primary growth occurred, and C02
evolution was greater in low nitrogen medium (14C labelled wheat seedlings in 56 mM
glucose, 2.4 mM NH4) than in high nitrogen medium (56 mM glucose, 24 mM NH4).
Still T. versicolor mineralized more 14C lignin in high nitrogen medium than either P.

chrysosporium or Sporotrichum pulverulentum (an asexual anamorph of P.
chrysosporium). Though the conversion of lignin to C02 was largely suppressed by

these high nitrogen conditions, the lignin was substantially solubilized by the white-rot

32

fungi in high nitrogen medium, up to 20% by P. clzrysosporium (65). This indicates that
while lignin mineralization may have been suppressed, lignin depolymerization under
these high ammonia, high glucose conditions may still have occurred. Low nitrogen
ligninolytic conditions as described above were also shown to induce T. versicolor to
produce enzymes capable of catalyzing the cleavage of 13-0-4 lignin model dimer
linkages (48).

These findings led to the discovery of extracellular peroxidases in T. versicolor
which had similar enzymatic properties as those found for the UPS and MNPs of P.
chrysosporium (21). The peroxidase activity could be induced in low nitrogen media
(2.2 mM NH4, 11 mM glucose) after seven days growth, or in carbon limited cultures
(10.2 mM NH4, 1.1 mM glucose) on day seven, but only if veratryl alcohol was added as
an inducer. The peroxidase isolated was a glycosylated 50 kd enzyme which could
catalyze the oxidation of veratryl alcohol, free phenolics, and the lignin model dimer 1,2-
di(3,4 methoxyphenyl)-1,3-propanediol which resulted in Cor-CB cleavage to yield
veratraldehyde. Surprisingly, oxygenation of cultures was found to be inhibitory to
enzyme activity. This is contrary to previous reports of increases in ligninolytic activity
upon oxygenation of T. versicolor cultures (77). The LIP (46) and the MNP (45) of T.
versicolor were isolated and characterized from day 11 carbon limited, stationary, non-
oxygenated cultures. Three LIP isozymes were isolated by anion-exchange
chromatography and chromatofocusing which ranged in size from 43,000 to 45,000
daltons and with pl values between 3.2 to 3.4. Several smaller peaks were also observed,
but not isolated. An MNP was also isolated and found to be around Mr 49,000. Both
sets of enzymes had enzymatic activities definitive for UPS and MNP3. The LIP
isozymes were found to oxidize non-phenolic B-0-4 and 13-1 lignin model compounds in
the presence of H202 and were, therefore, confirmed as LIP enzymes. Sequencing of the
[amino termini of the three isozymes revealed minor differences between them which

suggested the existence of several structural genes for UPS in the genome of T.

33

versicolor. The amino termini of all three isozymes were more similar in sequence to the
P. Chrysosporium amino terminus of LIP2 than to the amino termini of the other LIPs P.
chrysosporiwn (47).

T. versicolor is also known to have a glucose oxidase activity as seen in P.
Chrysosporium, and a laccase activity which P. chrysosporium does not have (64).
Laccase is a copper containing oxidase which catalyzes one-electron oxidations of
phenols to yield phenoxy radicals. Ultimately four electrons are used to reduce one 02
molecule to H20 during one catalytic cycle (55). Laccase has been implicated in lignin
degradation by T. versicolor, and has been shown to depolymerize lignin model dimers
(49, 66). The ability of T. versicolor to degrade the lignin component of wood
efficiently has spurred research on using this fungus as an alternative to P. chrysosporium
in the important industrial applications of biological bleaching of kraft hardwood pulp (2,
57, 58, 63), and the degradation of xenobiotics (67). In both applications T. versicolor
has proven to perform as well, and in some cases better than, P. chrysosporium.

The goal of the research presented in this dissertation is to characterize the
structure of the LIP genes of T. versicolor and to determine the environmental signals
which regulate the UP gene expression in this important white-rot fungus. A
comparison of the number of LIP genes present in T. versicolor to those in P.
Chrysosporium will indicate if the presence of multiple LIP genes is a common feature
among the lignin degrading white-rot fungi. The homology relationships between the LIP
gene(s) of T. versicolor and P. chrysosporiwn will provide valuable information on the
evolution of LIP multigene families, and may indicate the location of important
conserved regions within the amino acid sequence such as LIP active sites or substrate
binding sites.

Considering that both fungi utilize the same resources found in the lignocellulosic
material of wood, it is of special interest to find out how the LIP genes of T. versicolor

are regulated in comparison with those of P. cltrysosporium. Nitrogen and carbon

limitation have been shown to be general signals for induction of the ligninolytic system
in both P. chrysosporium and T. versicolor. Are the LIP genes of T. versicolor also
induced by nitrogen limitation in defined medium, and is there differential regulation
such that some of the gene transcripts are more abundant in the total RNA than others?
Most importantly which of the LIP genes produce transcripts when the fungus is grown
in wood and how does this compare with the major gene transcript(s) produced in
nitrogen limiting medium? The answers to these questions will indicate how well the
general signal of nitrogen limitation simulates the gene induction signals the fungus
responds to in wood, and if there are other as yet unknown environmental signals which
activate LIP gene expression.

Also both P. Chrysosporium and T. versicolor have been shown to degrade
hardwoods (angiospenn) faster than softwoods (gymnosperm). This has been related to
the type of lignin present in hardwoods (syringyl/guaiacyl) versus that found in
softwoods (guaiacyl). Furthermore, it has been shown that the rate limiting step in
guaiacyl lignin degradation is the depolymerization step, and that mineralization of the
depolymerized subunits of guaiacyl and syringyl/guaiacyl lignin occurs at equal rates
(26, 40). The differences between hardwood and softwood degradation rates may be
related to LIP gene expression induced by growth in either gymnosperm or angiosperm
wood. If that is so, then that may indicate that the general phenomena of differential LIP
gene expression is related to differences in the catalytic activity or substrate binding
ability of the various LIP enzymes coded for by the VLG genes of T. versicolor to the
widely varying types of lignin polymers present in nature.

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CHAPTER I

CIDNING AND CHARACTERIZATION OF A LIGNIN PEROXIDASE GENE
FROM THE WHITE-ROT FUNGUS Trametes versicolor

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Vol. 179, No. 1, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
August 30, 1991 Pages 428-435

CLONING AID CHARACTERIIAIIOI Of A LIGNIN PEROXIDASE GENE PRO“

II: warn-now unions Ira-sees versicolor

Andrew K. Black1 and C. A. Reddyl'z.

1Department: of Microbiology and Public health, and 2:18!” Center for

flicrobial Ecology, Michigan State University, Bast Lansing, MI. 48824-1101

Received July 19. 1991

 

Six putative lignin peroxidase (LIP) genes were isolated fros a Am
phage library of the white-rot fungus, Ira-etes versicolor, using the
Phanerochaete chrysosporius LIP cDNA (2.05 as the probe. Sequence analysis of
one of the genes, V1.61, showed that: its coding region is interrupted by six
small introns (49-64 bp) and that: it encodes a nature LIP protein (341 as: at:
36,114) that: is preceded by a 25 aa signal sequence. This protein has a
relatively high degree of as homology to the l—tersini of the LIP proteins
purified from 1'. versicolor and has an as honology of SS-60t to the LIP
proteins of P. chrysosporius, which is comparable to that. found between P.
chrysosporius and Phlebia radiaca LIP proteins. 0 1991 Academic Press, Inc.

 

Lignin peroxidases (LIPs), a family of sxtracsllul'ar, glycosylated, heme
proteins demonstrated in white-rot: fungi such as Phanerochaete chrysosporius,
Trametes versicolor, and Phlebia radii-ca, are believed to be important in
lignin degradation, as well as in degrading certain xenobiotics (1). Several
LIP cDNAs (2,3) and genes (reviewed in ref. 4) of P. chrysosporimn and one CD“
from Phlsbia radiate (5), have been isolated and characterized. However, there

S

has been no report: to date on the LIP genes of the over 1,600 species of other
wood~rotting fungi. .

Trametes versicolor, the next: best studied white-rot: fungus after P.
chrysosporium, produces several lignin peroxidases that, similar to the P.
chrysosporiura LIPs, are produced only during secondary metabolism in response

to nutrient starvation (6,7). Furthermore, antibodies against lignin

peroxidases of P. chrysosporium were shown to cross react with T. versicolor

 

0
Corresponding author.

 

Ll‘l'gp'c

47

Vol. 179, No. 1. 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

LIPs indicating the structural relatedness of LIPs from these two organises

(B). The objective of this study was to isolate and characterize LIP genomic
clones of 1'. versicolor and compare the sequence and other structural features
of one of the LIP genes with those of P. chrysosporium and the LIP encoding
cDNA of Pb. radiata.

IIaterials and Methods

Lignin pgngLdasg gene isolation and sgggencing. Genomic DNA of 1'. versicolor

(Coriolus versicolor) strain ATCC 12679 was extracted as described by Rae and
Reddy and a 1mm genomic library was constructed (10) which was screened
using P-labeled P. chrysosporium LIP cDNA CLGS as the probe (2,11). The
genomic fragments from the positive lambda clones were subcloned into pUClB and
p0C19 vectors and one of the LIP genes designated VLGI (see Fig. 1) was
sequenced as described previously (12), in both directions, using the dideoxy
chain termination procedure.

lesults and Discussion

- enom c es. Three AEHBL3 clones containing six
putative LIP-encoding regions were isolated. Probing of various restriction
digests of these three clones. with 32P-labeled CLGS cDNA showed that LIP gene
VLG! occurs alone on clone 1; genes VLGZ, VLGJ, and VLGC are located on clone
2; and genes VLGS and VLG6 are located on clone 3 (Fig. 1). The linked LIP
genes on clones 2 and 3 are separated by 1.5-2.0 kb DNA. Similar lihkage has
been reported for the LIP genes of P. chrysosporium (13,14). The results,

however, showed that the transcriptional orientation of the linked genes in T.

versicolor is unidirectional (Fig. 1) whereas in P. chrysosporium the

 

 

 

 

 

n91
...
...-II-
B SaP B
IIDKb
!L62 1LG3 2:64
--e -e -e
l----------- mum-mumm-
BSs SaSa B H 83 59B Sa s 9385: P
H P So P P 14 R P 53 51] So 'SESa SvSh
1 l 1 : l ' ' a H r I
VLGS VLGB
-e -e
—
K RKRSaSa H
P Sa Ss IR \S VSsJSaXSal S: P
L K 11 11 {‘1 J
Fig. 1. Restriction maps of the LIP-encoding genomic clones of T. versicolor.
The abbreviations used for restriction enzymes are 8, BamHI; R, EcoRI; H,
flindUI; K, Kpnl; P, Pstl; 5.), Salt; Sp, Spht; 55, 55:1; and x. Xba!;. The

boundary and the transcriptional direction of each gene are represented by a
dark box and an arrow, respectively. above each LIP gene.

48

Vol. 1 79. No. 1, 1 991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

transcription of linked genes, 190A and lpoB, was reported to be in opposite
directions (13).

Mence analysis a: 9&1. Complete sequencing of VLG! and aligning its
sequences with the LIP genes of P. chrysosporium (4) revealed an open reading
frame (our) of 1,098 bp that encodes 366 aa (Pig. 2). The VLG! on: was
identified by using the intron splice site consensus sequences compiled for P.
chrysosporium (see ref. 4) and the homology to the OR? of CLGS cDNA as guides.
The mature LIP protein encoded by VLG! is 341 as (at 36,714) and is preceded by
a 25 aa signal peptide which ends in the consensus proteolytic cleavage site
Arg-Arg. The N-terminal sequence of the V151 protein is 'very similar to the
experimentally determined N-terminal aa sequences of the lignin peroxidase
isozymes of T. versicolor (15), and the lignin peroxidase isozyme H2 of P.
chrysosporiue (2) shown below. This suggests that VLG! encodes a protein that
shares a high degree of homology to the known LIP isozymes of T. versicolor and

P. chrysosporium .

G" N RO BASE W
E. chgsosgrium H2 VAL-ALh-CIS-PRO-ASP-GLY-imL-flIs-THR-ALA-SER-ASN-m
I. vers icolo; A VAL-THR- 7 -PRO-ASP-GLI-LYS-hSN-THR-ALA-THR-ASII-m
I. vergigglo; B VAL-THR- 7 -PROsASP—GLI—VAL-ASN-THR-ALA-THR-ASlI-m
I. gggsicglog C vas-m- 7 -PRO-ASP-GLY—VAL-ASN-TRR-ALA-m-ASN-m
I. versicolo; VLG]. VAL-ALA-CYS-PRO-ASP-GLY—ARG-HIS-THR-ALA-TflR-ASN-ALA

Consistent with the fact that LIP proteins are glycosylated, the M1
protein contains one N-glycosylation site with the general sequence Asn-Xaa-
Thr/Ser (see Fig. 2), similar to that seen in P. chrysosporium LIP proteins.
In comparison to the VLGI-encoded LIP protein, the mature proteins encoded by
the three major LIP genes of P. chrysosporium each contains 344 aa and are
preceded by a 27-28 aa signal peptide that ends in the consensus Lys-Arg
cleavage site (see ref. 4). The LIP-encoding cDNA of Ph. radiaca on the other

hand encodes a mature protein of 337 aa and a 24 aa signal peptide which,

49

Vol. 179, No.1, 1991

GGCGCGACACACOGACGTGTCGGACGCTCCCGCGGGCACGCTGCACATGGTCGGCCCCGCTCGTGTTCCTCGGGG
TGGACAAAATACTTGCGTTGCGATTGGTCCGAGGCGCGGTTGCAACAGAGGACGGCTCACAATCCATTGTGGCGG
TCGTCCTCGGTTCCTTGAGGAGGGAGCCGGGATGAGCCCCGACATCGAGAGGGCCAGGAAATATAAAAGGTGGAC
GAAACGGCGCAGAACCTTCAGAGCACTCCAGCGTACTCCTCTCCCCTCTCTTCCACCTCCCTCGGCACCGGCAAC

ATC GTT TCC AAG TTC TTC ACC TCC CTC CTC TCC CTC GCT GCT GTC
Het Val Ser Lys Phe Phe Thr Ser Leu Val Ser Leu Ala Ala Val
G gtacgtggacgttgtttaaatcgtagtcgcctagctgatcctgttatag CT TCC
A IVS 1 la Ser
GTT CCC TCC CCC GAC GGC ACC CAC ACC GCT ACC AAC GCG GCT TGC
Val Ala Cys Pro Asp Gly Arg His Thr Ala Thr Asn Ala Ala Cys
CCT CTC CCC GAC GAT CTC CAG GCC AAC CTC TTC GAC CCC GGC AAC
Pro Leu Arg Asp Asp Leu Gln Ala Asn Leu Phe Asp Gly Gly Lys
CCC CAC GAG TCT CTC CCC TTC ACG TTC CAC GAC GCC ATC CCC ATC
Ala His Glu Ser Leu Arg Leu Thr Phe His Asp Ala Ile Ala Ile

 

CTC
Leu
CTC
Leu
TGC
Cys
TGC
Cys
TCG
Ser

GGT
Gly
ACC
Thr
GCT
Ala
AAC
Asn
CCG
Pro

GCT
Ala

CCC
Arg
CTC

Leu
GCT
Ala
CCC
Ala

AAC
Asn
CGT
Ass
TTC
Phe
GAG
Glu
CTC
Leu

GAG GCG CAG GGC AA gttcgggttagtggatacgcaatgcattgcagatcatcatcactcactagactac

Glu Ala Gln Gly As IVS 2

gcattacag C GGT GGA GGT GCC GAC GGC TCC ATC ACC ATT TTC TCG CAC ATC GAG ACC

n Gly Gly Gly Ala Asp Gly Ser Ile Thr Ile Phe Ser His Ile Glu Thr
GGC TTC CAC CCC AAC ATC GGT CTC CAC GAG GIT CTC GAG AAC CA6 COG CCT TTC CTC
Gly Phe His Pro Asn Ile Gly Leu Asp Glu Val Val Glu Lys Gln Arg Pro Phe Leu
CAC CCC CAC AAC ATC GGT GTT GCT GAC TT gtgagttgcacagcacgccccagggttttcacgggc

Gln Arg His Asn Ile Gly Val Ala Asp Ph IVS
tcccgctcatgcttcgttcacag C ATT CAA TTC GCC GGT GCC CTC GGT

GCA GGT GCT CCC CAG CTC AGC GCC TTC GTC GGC CCC AAC GAG COG
Ala Gly Ala Pro Gln Leu Ser Ala Phe Val Gly Arg Lys Glu Pro
CCC GAC GGC CTC GTC CCG GAG CCG TTC CAC ACC CCC GAC CAG ATC
Pro Asp Gly Leu Val Pro Glu Pro Phe His Thr Pro Asp Gln Ile
GCC CAC CCC TCC TCG GGC GAG TTC GAC GAG ATC CTC ACC GTC TGG
Ala Asp Ala Ser Ser Gly Glu Phe Asp Glu Ile Leu Thr Val Trp
CAC AOG ATC GCC GCC GCC AAC CAC GTC GAC CCC ACC GT6 CCC GGC
His Thr Ile Ala Ala Ala Asn Asp Val Asp Pro Thr Val Pro Gly
TCC ACC CCC GAG ATC TTC GAC TCG CAG TTC TTC CTC GAG ACC CAG
Ser Thr Pro Glu Ile Phe Asp Ser Gln Phe Phe Leu Glu Thr Gln
GCC TTC ACC GGG CCC GGC CCC GTG CAG GGC GAG GTC ACG TGC CCG
Ala Phe Thr Gly Arg Gly Pro Val Gln Gly Glu Val Thr Cys Pro
TTC CGC CTG CAG TCC GAC TTC GOG ATC GCG CCC GAC CAG GCC ACC
Phe Arg Leu Gln Ser Asp Phe Ala Ile Ala Arg Asp Gln Ala Thr
CAG TCG TTC GTC AAC AAC CAC ACC AAG GTC CAG CAG ATC TTC CAG
Gln Ser Phe Val Asn 555 Gln Thr Lys Val Gln Gln Het Phe Gln
CAC CTC TCC ATC CTC GGC CAG AAC ATC GAC CAC CTC GTT GAC TGC
Asp Leu Ser Ile Leu Gly Gln Asn Ile Asp Asp Leu Val Asp Cys
ctatacatttctcgtcagaggatgctcaacgatctgacttgttcttgtcgtag ATC
IVS 4 Ile
CCC CTC ACC ACC AGG ACC CAC TTC CCC GCC GGC ATC ACC CAC CCC
Pro Leu Thr Thr Arg Thr His Phe Pro Ala Gly Het Thr His Arg
GCT gtgagtcattcagttccattagacacttgccgtgctcacacatcctatcag ICC
Ala IVS S Cys
TTC CCC ACC CTC CCC ACC CAC CCC GGA CCC CCC ACC GOT CTC CCC
Phe Pro Thr Leu Pro Thr Asp Pro Gly Pro Arg Thr Gly Val Ala
cttcttcaactcacgaccgaccacaatctgaccgctcctccag C ATC CCC AAC
IVS 6 l Ile Pro Lys
GTAAACGGAGCAGCAACGCTCTCCCCGGCACACGGCTATCGGCGGTTCAGGATCC

Fig. 2. Complete nucleotide sequence of T. versicolor LIP gene VLG!
accession number: “55294) and the deduced amino acid sequence.

Putative TATA box and CAAT box, signal peptide cleavage site.

3
CCC

ACG
Thr
TTC
Phe
CTG
Leu
TCG
Ser
CTC
Leu
TGC
Cys
GCG
Ala
TTC
Phe
ACC
Thr
CCC
Pro
CAC
Asp
TTC
Leu
CCC
Pro
CGG
Arg

TCC AAC TGC
e Ile Gln Phe Ala Gly Ala Leu Gly Ala Ser Asn Cys

CCC
Arg
GCC
Ala
CTC
Leu
CCG
Pro
AAG
Lys
CCC
Ala
TGC
Cys
CTC
Val
GAA
Glu
ATC
Ile
ATC
Ile
GAG
Glu

CCC
Pro
CCC
Arg
ACC
Thr
TTC
Phe
GGC
Gly
GGC
Gly
GAG
Glu
TTC
Phe
CTC
Val
CCC
Pro
GAG
Glu
ACC
Thr

GCC
Ala
ATC
Ile
GCG
Ala
GAC
Asp
ACC
Thr
GAG
Glu
TGG
Trp
CAC
His
gta

AGG
Arg
CAC
Gln
CCC
Pro

GT gtaagtct

Va
CTC

TAG

Val Stop

(EMBL

The nucleotide
sequence of the introns (IVS! to IVS6) are given in lower case
and putative N-
glycosylation sites are given in bold face letters and underlined.

letters.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

75

150

225

300

357

424

481

538

595

664

722

779

B44

904

961

1018

1075

1132

1189

1246

1303

1360

1417

1483

1540

1611

1669

1731

1786

50

Vol. 1 79, No. 1, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

similar to that of T. versicolor LIP protein VLGl, ends in the putative
proteolytic cleavage site, Arg-Arg (5). '

The codon usage for the LIP protein encoded by VLG! is extremely biased in
favor of codons ending in C (6“) or G (30t). Thus, 97‘ of the total codons
used for the VbGl LIP protein end in a C or G. In LIP proteins of P.
chrysosporium also, the codon usage is heavily biased in favor of the codons
ending in a C or G (2).

The ATG initiation site (Pig. 2) is located within the consensus
eukaryotic initiation sequence A/GIIIIATGG (16) and is very similar to that found
in the genes encoding LIP isozymes H2, H8, and H10 in P. chrysosporium (see
ref. 4). Putative TATAA box and CAAT box, the consensus eukaryotic promoter
elements, are, respectively, located 80 bp and 165 bp upstream of the ATG
initiation site.

a id h l to the LI roteins of other white- at fun . A comparison
of the as sequence of the LIP protein VLG! to that of P. chrysosporium LIP
isozymes 32, as, and mo (Pig. 3a, showed. sv-sn homology, whereas the a
homologies are much higher among the latter three LIP isozymes of P.
chrysosporium (70-80t). The LIP protein VDGl shares 60‘ homology to the LIP
protein LIII of Pb. radiate.

It has been well established that in a variety of peroxidases, including
the turnip peroxidase, cytochrome c peroxidase, horseradish peroxidase, LIP
isozymes of P. chrysosporium. and Pb. radiate LIP protein, a proximal histidine
(which serves as an axial ligand of heme) and a distal histidine and arginine ‘
(which are involved in charge stabilization during reaction of the heme with
H202) residues are well conserved. These critical aa residues and the as
sequences surrounding these residues are also highly conserved in VLG! encoded
LIP proteins (Fig. 38).

Introns. The coding region of VLGI is interrupted by six relatively small
introns (size range 49 to 64 bp), including one intron (IVS!) which interrupts
the signal peptide coding region (Fig. 2 and Fig. 4A). In comparison, the LIP
genes of P. chrysosporium described to date contain eight to nine introns and

the positions of these introns are different from those in VLG] (Fig. 4A).

lie

51

179, No. 1, 1991

A
1 10 20 30 40 SO 60

NVSKPPTSLVSLAAVLGAIASLIA---IVACPDGRHTATIAACCALPPLRDOLOAILPDGGKCRAIAHI
AP QLLAAL V L? QVTOAAPILDK V S H O 6

AP QLPAAI LL 8 AIAAAVIIK AT 8! K- VCD 8
AP KLLAVLTA L8 P AOGAAVIK- A! 8' KVVPA- 8
APOLLAI‘LAAS-V---IA? ”LII-B

VLBI
C164
“Ll

CLGS
IBPJ

H VL
H DVL
TV IVLS
LAV

-10 - so so 100 110 120 130 140
mmaxspwx-mmmxmmxcwsvmoapmxcvannormmx
a xv s a as xrc IT r! A: x xxx cvrns a v v

r v 's x xrs ' a x xi x v x cvrss a vaL
x v x s assvx— s 1 IL x v x cvrss a van
x

8
DD
08
A N 1 0’0 0 8P 0 SCH V T

150 160 170 100 190 200 210 220
CIGAPOLSAPVCRKZPTIPAPDGLVPIPPHTPDQIPAIIADASSGBPDBILTVHLLTAHTIAAANDVDPTVPGSPPDST
IQ! L P A CA I VL IL - 6 IS L
HI? I APA 0 II VI - V O L
HIP T APA Q ID V? '10

I DA CA L P! A SEA

1 I

'0'.“
H1001”
I"

222"

I
LIL
LZL
L!

Q I I
H n

V
8V
DVIT 9

230 240 250 260 270 280 290
PBIWSQPWWWMWWWPAIWTACWWWU IL

cg v r HLP sa 1 TA r
c v 1 LA to
v
I

I H 2
a

SR
6 L AR I LA 19
SVH 6 IR I IAHGQ
310 320 330 340 350
GQIIDDLVDCTIVIPIPRPLTTR----THPPAGHTHIDIDQACLBTPPPTLPTDPGPRTOVAPVIPKRVO
HDHNAKI 8 A I VIPGP---SP A RIP PPSPNO
DPNAKT 80 05K IPGNLPP-SP K GRIP PPGAO

S
DP ANT 8A SK APNNTPGPSP P 8 RIP PPGAO
TDPTT I SD L V P S V----P I A A PAD--

E

K

K I
ND
II

<<<

i??:
a d H
or,

P’H

P

VLGI
82
38
H10
LIII
1P
CCP
KRP

S6

56"""163
55......lszccrnrxrrvuttsa

SS......IGIGBPDELZLVHHLSA

163 H
H
H
......IGIGEPDELELVHHLSA 8
a
H
a
H

11mg;
SIAAAN182
SVAAVIII81
SVAAVI181
SVAAQN17‘
TIGQSRlBO
ALGKTHI76
TICKNQ

Pig. 3. conservation of amino acid sequences between the LIP proteins of P.
cbrysosporium, Pb. radiate, and VLGl of T. versicolor. A. Amino acid homolgy
between the LIP protein encoded by T. versicolor gene VLG] and those encoded by
the LIP coma. cscc, eras, ana'nnx of p. cbrysosporiun and LGPJ of Pb. radiate.
CLGl, KL), and CLGS are LIP cDNAs of P. cbrysosporium that, respectively,
encode LIP proteins, H2, H8, and H10 (2,3). LGPJ is a LIP cDNA of Pb. radiata
that encodes the LIP protein LIII of this organism (5). Proximal and distal
histidine and arginine residues known to be required for catalytic activity of
peroxidase proteins are marked with an asterisk. 8. Comparison of the active
site regions of the LIP protein VLCl of T. versicolor. LIP proteins H2, H8, and
1110 of P. cbrysosporium (2,3), LIII of Pb. radiata (S), turnip peroxidase,
cytochrome c peroxidase, and horse radish peroxidase.

LTP DAIAISPA
DSIAISPX
DSIAISPA
DAIAISPAS‘
DAIAISPASO......15‘
DCPVIIGC061 16OVCLSTRDHVALSGA
TSGT‘DKHSO......ISGRLNHDREVVALHGA
DCPVIGCD

GLNRSSDLVALSGC

GEPDBILTVHLLTA

LVP
LVP
LIP
LPP
LAH
LHP

GDPDZLELVUPLIA

.00...

g

However,

internal

ENCKJHEIAKJALAAAMJIBK)PIFHSKJAL.RE§NBARKJI(JOMAAJLHUKJAJICHVS

the consensus exon/intron splice junction sequences and the conserved

sequences

of

VL61

N/GTRNGT......CTSAY ...... YAG/Y,

(Fig.

48)

are

similar to those seen in the LIP genes of P. chrysosporium and other eukaryotic

52

 

 

 

Vol. 179. No. 1. 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
* zoo-p
11.9.! -' — - -_J
113. — _-__—_ —_-_a
m ..L—Jl -"-‘ —"

B

n51auxAnaanoouxrmnrumwnxnmmaxtnw --------- cuss -------- ccmnnumcnn
n52(mAflnTdchnmnomumammxnxmrnxucxnn:data«ntacdmounnaxmrnuuqax
IVS3 CTT/GTCAGTTCCACAGCACGCCCCAGGGTTTTCACGCGCICCCG--CTCAI ----- CCTTCGTTCACAG/CA
IVSA CTC/CTAcTAIACAI1TCTCGTCAGAGCAIGCTCAAGGAI ------- CTCAC-o-ITCTTCITCTCCTAG/A1
IvssccnflntMntarumsrnxmrnmaawmuxxcn: --------- (TOM: ------ AQUCCUUCMVTG
IVS6 CGT/CIAAGTCTCTTCTTCAACTCACCACOGACCACAAI -------- CTCAC ------- CCCTCCTCCAC/CA
T. versicolor HK/CTRHGT 34-41 CTSAY 10-17 VAC/Y

P. chrysosporium SKICTRNGY 32-37 YTCAY 9~l9 YAG/H
Higher Eukaryotes CAG/GIAAGT CTAAT “CAC/G
Yeast l/GTAIGT TACTAAC HYAC/H
Filamentous fungi ‘IGTAYCTT ICCTAAC ACAG/g

Pig. 4. comparison of the intron positions and the splice junction sequences
in the LIP genes of T. vorsicolor and P. chrysosporium. A. A comparison of the
position of introns in the T. versicolor LIP gene VLG! and P. cbrysosporium
genes which encode LIP proteins s2. 88. and H10 (from ref. 17). Closed boxes
represent exons and open boxes represent introns. D. Conserved exon/ intron
junction sequences and internal conserved sequences of introns of. the VLG!
gene. The boundaries of exons and introns are marked by slashes.
Abbreviations for single letter codons are: H, A or C or G or T; R. A or G: I,
C or T: S, C or G. The conserved intron/exon junction sequences and internal
conserved sequences of P. cbrysosporium LIP genes (4), and of genes of higher
eukaryotes, yeasts. and filamentous fungi (16) are also presented.

genes suggesting that these genes share similar splicing mechanisms (4,16).
The sizes of the introns in VLG) and LIP genes of P. cbrysosporium are also

very similar.

ACKNOWLEDGMENTS

He wish to thank Dr. Prischauf of the European Molecular Biololgy
Laboratories, Heidelberg, Germany for supplying us with the lambda vector EMBL3
and with its host strains Escherichia coli HMS38 and NH539. This work was
supported in part by the Michigan Agricultural Experiment Station. and grants
DE-FGOZ-BSER 13369 from the U. s. Department of Energy and lROl-GM39032 from
NIH.

REFERENCES

1. Kirk, T. K. and Farrell, R. L. (1987) Ann. Rev. Hicrobiol. 41, 465-505.

2. deBoer, H.A.. Zhang, 1.2.. Collins. C. and Reddy, C.A. (1987) Gene 60,
93-102; Corrigendum (1988) Gene 69, 369.

3. Tien. M. and Tu, C-P. D. (1987) Nature 326. 520-523.

4. Zhang, Y. 2., Reddy, C. A., and Rasooly, A. (1991) Gene 97, 191-198.

S. Saloheimo, M., Barajas, V., Niku-Paavola, M.-J. and Knowles, J.K.C.
(1989) Gene 85, 343-351.

6. Dodson, A. P. J., Evans, C. 5., Harvey, P. J. and Palmer. J. H. (1987)
FEMS Microbiol. Lett. 42, 17.

7. Jonsson, L., Johansson, T., Sjostrom, K. and Hyman, P. 0. (1987) Acta

Chem. Scand. 41, 766-769.

Vol

10.

11.

12.

13.

14.

15.

16.
17.

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Garcia, 8., Latge, J. P., Provost, M. C. and Leisola, M. (1987) Appl.
Environ. Microbiol. 53, 2384-2387.

Rae, T.R. and Reddy, C.A. (1984) Biochem. Biophys. Res. Comm. 118, 821-
827.

Sambrook, J., Pritsch, 8. P. and Maniatis, T. (1989) Molecular Cloning.
A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, lew York.

thang, I. 8. and Reddy, C. A. (1988) Methods lnsymol. 161, 228-237.

Zhang, H., Scholl, R., Browse, J. and Somerville, C. (1988) Hucl.
Acids. Res. 16, 1220-1221.

Huoponen, K., Ollikka, P., Kalin, M., Walther, 1., Mintsfili, P. and
Reiser. J. (1990) Gene 89, 145-150.

Raeder, 0., Thompson, H. and Broda. P. (1989) Mol. Microbiol. 3, 911-
918.

ansson, J., Karlsson, 0., Lundquist, K., and Hyman, P. 0. (1989) PEBS.
Lett. 247, 143-146.

Ballance, D. J. (1986) Yeast 2, 229-236.

Naidu, P. S., thang, Y. z. and Reddy, C. A. (1990) Biochem. Biophys.
Res. Chem. 173, 994-1000.

CHAPTER II

Structure and Molecular Regulation of VLGZ,
a Li gnin Peroxidase Gcnc From

the White-Rot fungus T rametes versicolor.

54

55

ABSTRACT

Transcripts (1.25 kb) of the lignin peroxidase gene VLGZ from the white-rot
fungus T. versicolor were detected in a nitrogen limited medium (56 mM glucose, 2.2
mM NH4) containing low levels of Mn(II) (0.13 ppb). The presence of transcripts of the
other 5 LIP genes cloned from this fungus could not be confirmed under the above
conditions. At moderate Mn(II) levels (2.6 ppm), LIP activity of T. versicolor was
completely suppressed. A cDNA library was prepared, screened, and VLGZ-spccific
cDNA (designated VLCZ) was isolated Sequencing of both VLGZ and VLCZ revealed
that VLCZ is 79% homologous at the nt level and 76% homologous at the a level to the

f

coding region of VLG] .

56

INTRODUCTION

Woody plant tissues are made up of three major structural polymers: cellulose,
hemicellulose, and lignin. The lignin component makes up 20-30% of the dry mass of
woody plants, and is the most abundant renewable aromatic polymer on the planet (32).
Because it is a random polymer of phenylpropanoid subunits that is chemically
associated with cellulose and hemicellulose in the cell wall, lignin provides physical
rigidity to plants as well as a barrier to fungal and bacterial plant pathogens. White-rot
fungi are among the few organisms capable of mineralizing all the three components of
wood (4).

Interest in these organisms has increased since the discovery of lignin peroxidases
(LIP) and manganese peroxidases (MNP), which are the key enzymes responsible for the
initial depolymerization of the lignin polymer (12, 26, 37, 38). LIPs catalyze H202-
dependent one electron oxidations of both the phenolic and non-phenolic groups in the
lignin polymer (13, 21, 35, 36). MNPs catalyze one electron oxidations of Mn(II) to
Mn(III) which then oxidizes the aromatic carbons in the lignin (11, 41). These enzymes
can be induced by either carbon or nitrogen starvation during secondary metabolism (9).

The lignin peroxidases of the white-rot fungus P. chrysosporium have been the
most well studied. Multiple LIP isozymes have been isolated and cDNA as well as the
genomic clones encoding these isozymes have been characterized (6, 23, 28-30, 39, 40,
43). These studies have shown that each LIP isozyme is encoded by a distinct gene of the
UP multi-gene family (5). The LIP genes are reported to be clustered within the genome
of P. chrysosporium (31), but only two such clusters have been identified to date.

The LIP isozymes are differentially regulated such that under nitrogen limitation

at least six readily identifiable LIPs (designated LIPl-LIP6) are induced, but three

57

predominate (23): LIP2, LIPS and LIP6. A study of the molecular regulation of UP
genes has shown that the levels of expression of LIP isozymes is correlated with the
levels of the corresponding LIP RNA transcripts (4, 15).

T. versicolor has also been shown to produce multiple LIP isozymes (18, 19) and
a single MNP isozyme (17) under both nitrogen and carbon limitation conditions (7).
We have previously described the cloning of a family of UP genes from the white-rot
fungus T. versicolor (1). We present here the sequence analysis of the second LIP gene,
(VLGZ) of T. versicolor. Furthermore, since T. versicolor has been shown to mineralize
lignin and express LIP activity under conditions of nitrogen or carbon starvation, we
studied the molecular regulation of the six LIP genes cloned from T. versicolor. We
present evidence that under nitrogen limited conditions, T. versicolor produces an
abundance of VLGZ transcripts, and that LIP activity of T. versicolor is regulated by

Mn(II) concentration in the medium.

58

MATERIALS AND METHODS

Culture Conditions. T. versicolor (ATCC 12679) was used for all experiments.
The strain was maintained at 4° C on modified malt extract plates (2% malt extract, 2%
glucose, 0.1% peptone agar, pH 4.5). Modified low nitrogen medium (LN medium) of
Kirk et a1 (23) was used. This medium contained the following: Vitamins per liter: 2 pg
biotin, 2 pg folic acid, 5 pg thiamine, 5 pg riboflavin, 10 pg pyridoxine, 100 pg
cyanocobalamine, 5 pg nicotinic acid, 5 pg DL-calcium pantothenate, 5 pg p-
arninobenzoic acid, and 5 pg thiotic acid. Except where mentioned otherwise,
MnSO4.H20 was added to a final concentration of 400 pg per liter (0.13 parts per
billion) and Tween 80 was added to a final concentration of 0.001%. Four plugs of T.
versicolor were added from malt extract agar plates to 50 ml of LN medium without
Tween 80 in a 2.8 L Fernbach flask and grown at 25° C for 7 days. The culture was
homogenized and the homogenized inoculum was added to 10% of the final culture
volume (10 ml of low N medium in 125 ml of sterile foam-plugged Erlenmeyer flasks)
and then incubated at 25° C under static conditions.

Enzyme Assays and Analytical Assays. LIP and MNP assays were performed
as described previously (36). Glucose was measured as the reducing sugar by the
dinitrosalicylic acid method, using D-glucose as the standard (42). Mycelial dry weight
was determined by vacuum filtration of the mycelial mass onto tared Whatrnan #1 filter
disks, dried to a constant weight at 80° C, and weighed. Ammonium ion concentration
was determined by using an ammonia electrode (Fisher model 13-299-96, Fisher
Scientific Company, Pittsburg, PA) per manufacturers specifications.

mRNA Extraction and Northern Blots. Total RNA was extracted as described
by Haylock and Broda (14), and the poly(A) RNA fraction was purified by oligo-dT

59

cellulose (Bethesda Research Laboratories, Gaithersburg, MD) chromatography. 2 pg of
this RNA was electrophoresed on a 1.2% agarose gels with 2.2 M formaldehyde as
previously described (33). RNA was transferred to nylon membranes (Micron
Separations Incorporated, Westborough, MA) by capillary transfer and immobilized by
UV crosslinking (Stratalinker 1800, Stratagene, La Jolla, CA) at 400 J/m2. The blot was
hybridized with a 32P-dcrp labelled DNA probe containing the complete coding region
of VLGZat 42° C in 50% formamide, 1.0 M NaCl hybridization buffer.

cDNA probe preparation. cDNA probes were prepared as described by
Sambrook et al. (34). In brief, a poly-dT 17 bp primer was added to poly(A) purified
RNA and annealed at room temperature. The primer was elongated by addition of
MMLV-reverse transcriptase (BRL), and labelled with 32P-dCl‘ P added along with the
other nucleotides. The resulting single stranded cDNA probe, representative of the
mRNA species present in the RNA sample, was hybridized to a Southern blot containing
the VLG], VLGZ, VLG3, VLG4, VLGS, and VLG6 LIP genomic clones of T. versicolor.
The genes occured in linked sets on lambda clones as previously described (1). To
separate the genes, the lambda clones were digested with different enzymes. Clone 1
containing VLGZ, VLG3, and VLG4 was digested with EcoRI and HindIII; clone 2
containing VLGS and VLG6 was digested with PstI; and clone 3 containing a single gene,
VLG], was digested with BamHI. The digested clones were electrophoresed on a 0.7%
agarose gel, blotted to nylon and immobilized as above. The Southern blot was
hybridized to the cDNA probe at 48° C in 50% formamide and 400 mM NaCl
hybridization buffer; washed extensively at 68° C in 0.1 X SSC, 1% sodium dodecyl
sulphate; and an autoradiogram was prepared.

cDNA Library Construction. 5 pg of poly(A) purified RNA extracted from day
9 cultures grown in LN medium was used to construct a lambda phage cDNA library in
the vector gt22A using the Lambda Superscript system (BRL) as described by the
manufacturer. The library consisted of 1.4 x 106 phage clones; 50,000 of the phage

clones were screened on a 150 x 15 mm petri plate by immobilization of the plaques onto
nitrocellulose filters and hybridization to a 32P-dC'I‘ P labelled VLGZ probe. A lambda
cDNA clone, which hybridized to the VLGZ probe, was purified and the cDNA insert
was subcloned out of the EcoRI, NotI site on the purified lambda clone into the EcoRI,
SmaI site of pUC19. The insert was verified as VLC2 by sequencing 200 bp from its 5'
end and comparing it with the corresponding VLGZ DNA sequence.

Sequencing of VLGZ and VLC2. A 3.8 kb HindIII fragment from the lambda
genomic clone 1 containing the complete coding sequence of VLGZ was subcloned into
M13mp19 in both orientations. The complete 3.8 kb fragment was sequenced by
generating ordered deletions with nucleases EonI and SI (34). VLC2 was sequenced

similarly in both directions by using appropriate restriction fragments.

61

RESULTS

Nitrogen limitation. Growth of T. versicolor in low nitrogen medium (initially
2.2 mM NH4) leads to nitrogen depletion after 5 days, whereas glucose levels are non-
limiting through day 13 (Fig. 1). Biomass continues to increase in the cultures, even
after nitrogen depletion, until day 7 when biomass increase levels off and LIP activity
appears.

Lignin peroxidase enzyme induction and appearance of transcripts. Low
levels (0.13 ppb) of Mn (II) allowed relatively high levels of activity while higher levels
were inhibitory and at 2.6 ppm LIP enzyme production was completely suppressed
(Table 1). Measurable LIP enzyme activity appeared on day 7 (Fig. 2) under nitrogen
limiting conditions. Manganese peroxidase (MNP) activity appeared earlier than LIP
activity with peak values of 101.5 units/liter occurring on or before day 5 and decreasing
to 19.23 units/liter by day 11. To determine the temporal production of LIP transcripts vs
LIP enzyme activity, RNA was extracted from the samples on days 5, 7, 9, and 11. The
poly(A) RNA fraction was purified and a northern blot was prepared, (Fig. 2).
Hybridizing the blot to the VLGZ probe indicated that a low level of VLGZ transcript
(1.25 kb) was present on day 5 which correlated well with the onset of nitrogen
limitation on day 5. Transcript levels rose dramatically to peak levels on days 7 and 9,
and then fell somewhat by day 11. A positive correlation was observed between LIP
enzyme production and the level of VLGZ transcripts although there was a time lag
(about 24 hours) between the transcript levels and the translation of these into LIP
activity levels. The corresponding MNP transcript levels in the above cultures were not
determined since MNP genes have not yet been cloned from T. versicolor. Carbon

limited and nitrogen-sufficient medium, previously used to induce LIP enzymes in T.

62

versicolor strains PRL 28A (6) and PRL 572 (17), was tried with strain ATCC 12679 at
0.13 ppb Mn(II); however, no measurable LIP enzyme activity was seen under these
conditions.

Major LIP gene transcripts under nitrogen limitation. Six LIP genes have
been cloned previously from the T. versicolor genome (1). Some of the genes are tightly
linked within the genome (VLGZ, VLG3, VLG4, and VLGS, VLG6) with 1.5-2.0 kb
separating them (1). To determine which of the poly A RNA transcripts from these
genes was most abundant under nitrogen-limited conditions, a cDNA probe (see
materials and methods) was prepared from day 9 mRNA, and hybridized to the cloned
LIP genes immobilized on a southern blot. High stringency hybridization and washing
conditions were employed to minimize cross-hybridization between related LIP cDNA
probes. The resulting autoradiogram (Fig. 3) clearly showed that VLGZ is actively
transcribed under nitrogen limiting conditions. Similar results were obtained using day 11
mRNA. The second and third most intensely hybridizing genes were VLG] and VLG3,
respectively. However, at least for VLG], this hybridization must be due to cross-
hybridization since VLG] specific transcripts could not be found when northern blots
were probed with a 5' VLG] specific gene probe, nor could any cDNA clones specific for
VLG] could be found in the cDNA library constructed from day 9 mRNA (data not
shown).

Isolation of VLC2 and sequence analysis of VLGZ. A cDNA library was
constructed from the day 9 mRNA and a cDNA clone, which corresponds to the LIP
genomic clone VLGZ, was isolated. Both clones were sequenced and the intron/exon
junctions located. Fig. 4 contains the sequence of VLGZ with the intron sequences in
lower case letters. The amino acid sequence of VLGZ was deduced from the open
reading frame provided by VLC2 and placed below the corresponding DNA coding
sequence. A consensus TATAA box precedes the ATG translation initiation codon by 83

base pairs. Translation of VLGZ from this ATG results in a 368 residue protein. A

63

consensus signal sequence of 26 residues ending at the proteolytic cleavage site of Arg;
Agg (underlined and in boldface in Fig. 4) is located at the N terminus of the protein, and
is very similar to the signal sequence found in VLG] (l). Cleavage of the signal
sequence results in a 342 amino acid mature protein of Mr 36,075. Two potential N-
glycosylation sites of the general sequence Asn-X-Thr/Ser are located within the mature

protein at residues 103 and 214.

DISCUSSION

Nutrient limitations such as nitrogen or carbon starvation have been shown to be
necessary for induction of lignin degradation and lignin peroxidase enzyme production in
the white-rot fungi P. chrysosporium and T. versicolor (7-9, 20, 22, 25, 27). Lignin
model compounds, or the white-rot fungal secondary metabolite veratryl alcohol, are also
known to induce LIP enzyme levels (9, 10). Moreover LIP isozymes of P.
chrysosporium have been shown to be repressed in nitrogen- or carbon-limited media in
the presence of 2 8 ppm Mn(II) (3).

The results of this study showed that nitrogen—limiting culture conditions are a
necessary pre-requisite for the appearance of LIP enzyme activity in T. versicolor, but
only in the presence of very low levels of Mn(ID in the medium. The level at which the
LIP enzymes of T. versicolor were repressed (i.e. 2.6 ppm) is lower than the reported
range of 5-200 ppm Mn(II) present in wood (2, 3). However, the actual percentage of .
this manganese available to the fungus is not known since the Mn(II) may be bound to
wood in insoluble complexes.

The results showed good correlation between LIP enzyme levels and the level of
the VLGZ transcripts. VLGZ transcripts only appear when nitrogen has been depleted
from the culture. The observed correlation between the appearance of VLGZ transcripts
and the appearance of LIP enzyme activity is similar to that observed for LIP2 and LIPS
transcripts and the corresponding LIP isozymes H2 and H8 in P. chrysosporium (15). It
is possible that other LIP genes exist in this strain of T. versicolor that have not yet been
described, and that these genes are also induced under these conditions. It is also
possible that VLG] transcripts are present, but at levels too low to be detected by present

molecular techniques.

65

As the DNA hybridization results predicted, VLGZ has strong nucleotide
homology to VLG] and the protein sequences deduced from VLGI and VLGZ sequences
have a high degree of amino acid homology (1).

Amino Acid Homology Amongst The LIP Enzymes
VLGZ VLGl LIP2 LIPS LIP6 LGP3

VLGZ 76% 63% 61% 58% 62%
LIP2 72% 66% 58%
LIPS 82% 61 %
LIP6 58%

In fact when comparing the homology of the LIP enzymes of T. versicolor to that of the
other white rot fungi it is obvious that VLGl and VLGZ are more closely related to each
other than either is to the LIP enzymes from P. chrysosporium, or the LGP3 enzyme
from the white-rot fungus Phlebia radiata (33). VLGZ, next to VLGl, is most
homologous to the LIP enzyme LIP2 of P. chrysosporium. and LGP3 of P. radiata.

The molecular regulation of the LIP genes of T. versicolor and P. chrysosporium
is quite similar. Nitrogen and Mn (II) limitations are common environmental signals for
the appearance of the LIP transcripts of T. versicolor. Also, nitrogen limitation leads to
increases in transcript levels for some of the LIP genes more so than for the others, as
shown here for the increase in transcript levels of the VLGZ gene.

The presence of LIP multi-gene families in both T. versicolor and P.
chrysosporium with differential expression of the LIP genes under nutrient limiting
conditions raises the question of what the function of the multiple LIP genes is. Defined
low nitrogen media simulates some of the natural conditions the fungus encounters in
wood. Wood has a low nitrogen content and therefore white-rot fungi probably exist in a
constant state of nitrogen limitation when subsisting on wood. Therefore, it would be of
interest to determine if the same LIP genes are induced in natural substrates such as wood

as are induced in defined nitrogen limiting media.

10.

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TABLE 1. Effect of Mn(II) concentration on lignin peroxidase activity of T. versicolora

 

 

Mn(II) concentrationb Li gnin Peroxidasec
(parts per billion) activity (Units/Liter)
0.00 7.40
0.13 13.10
13.00 8.36
2600.00 0.00
a) T. versicolor was grown in low nitrogen media as described in Materials and

Methods. LIP and MNP activities were measured in 11 day old cultures.

b) Mn(II) was added as MnSO4.
C) LIP activity was measured as described (35). One unit of LIP activity represents

1 pmol of veratraldehyde produced from veratryl alcohol per minute.

71

FIG. 1. Nitrogen depletion versus lignin peroxidase production during growth of
T. versicolor in nitrogen limiting medium. Filled diamonds (0) represent glucose
concentration (mM); open circles (0) represent ammonium ion concentration
(mM); filled circles (0) represent biomass (dry weight in mg/flask); and crosses

(X) represent LIP activity (Units/Liter) of T. versicolor.

72

 

+aromm —O—Ammonh (mu) —-)(—-up (Units/um) +elueoe. (mM) *

 

 

 

(mg/flask)
12 -- n. 70
1o 1:- 4- w
1- 50
3 ..

Blames: (mg/flask) - - 40
Ammonia (mu) 6 <- Glucose (mM)
UP (Units/Liter) i' 30

4 4'
u- 20
I. .
2 . 1. 1o
0
0 ‘ '3'" ‘3‘ O O O r- 0
0 1 3 5 7 9 11 13

Days

73

FIG. 2. VLGZ transcript levels on different days of incubation. T. versicolor was
grown in low nitrogen media as described in Materials and Methods. LIP activity
(Units/Liter) was determined as described in the footnote of Table 1. Lanes 1
through 4 in the Northern blot correspond, respectively, to poly(A) RNA from
cultures on day 5, 7, 9, and 11. The blot was hybridized with a 32P-dCTP
labelled DNA probe containing the complete coding region of VLGZ at 42° C in
50% formamide, 1.0 M NaCl hybridization buffer.

75

FIG. 3 Concentration of transcripts of VLG], VLGZ, VLG3, VLG4, VLGS and
VLG6 in poly(A) RNA from day 9 cultures grown on low nitrogen medium. The
Southern blot was hybridized with a cDNA probe prepared from a day 9 poly(A)
fraction of RNA (as described in Materials and Methods), and washed under high
stringency conditions. Lane 1 contained EcoRI and HindIII digested DNA from
lambda clone 1 which contained the LIP genes (VLG4, VLGZ, VLG3); lane 2
contained DNA from lambda clone 2 (VLG6, VLGS) digested with PstI; lane 3
contained DNA from lambda clone 3 (VLG!) digested with BamHI.

76

123

 

 

77

FIG. 4. Nucleotide sequence of VLGZ and the deduced amino acid sequence of
the protein encoded by this gene. 5' upstream, 3' downstream, and exon
sequences are in upper case letters intron sequences are in lower case letters. The
first 6 nucleotides from the 5' end of VLC2 are in boldface. The sequence of
VLGZ ends at the polyadenylation site for VLC2. I

78

CCTACGTTCACAGTAGGTTACGGCAGCGATACAGCTTGGGCACACGAACTAGGATGGCCCTGGCGGGGAA 70
GGATCAGGGCGATGCTACAGCTCGCCGCCATTGGTCGCCTTGCACAACGTCACCGCCACACCAGAGTTGT 140
GGACCTCTCGCTAGTGCGCTTCATGGCGGCAAGGTTGTGAACGGCACCGAGAATGGTGGAATGGCAGAGC 210
TGCCATTGGGTCCTTGTGTCGATCTGTGCATCACAGCTCGGCTACCTATCCTTGGTCGCGGAGAGCCGGT 280
GAGGACCTTGCAGAGACACCGCCCAGTTTATTCACTATTTTCGAGGTTCACGACAGATATAAAAGAGGTG 350
ATGCGGCAGCCCAAAGACCTCAGAGCACTCGAGTTCTCCTCTCTTCGCCTCCCAACAGCTCGGCACCGAC 420
ATG GCC TTC AAA ACC TTA CTC TCC ATC GTC TCC CTT TTG GCG GCG TTC CAG GGC 474
Net Ala Phe Lys Thr Leu Leu Ser Ile Val Ser Leu Leu Ala Ala Phe Gln Gly

GCG ACC‘G«gtacgtggctagcgcggccgacctcgacatcatttgtcacgacaatctgacgtcttttcat. 542
Ala Thr A
ccgctag CC

ACT
Thr
CTG
Leu
TTC
Phe

AAC

la
GCG

Asn Ala
TTC CAC
Phe His

CAC
His

GAC
Asp

GCA
Ala
GCG
Ala
GGC

TTG
Leu
TGC
Cys
GGT

ACC CGC CGC GTC GCT TGC CCC GAC GGA GTG AAC ACC GCG 596
Thr Arg Arg Val Ala Cys Pro Asp Gly Val Asn Thr Ala
TGC CAG CTC TTC GCT GTG CGC GAC GAC CTC CAG GAG AAC 650
Cys Gln Leu Phe Ala Val Arg Asp Asp Leu Gln Glu Asn
CTC TGC ACG GCC GAG GCG CAC GAG TCT CTT CGC TTG ACG 704

Gly Gly Leu Cys Thr Ala Glu Ala His Glu Ser Leu Arg Leu Thr

GCT
Ala

ATC
Ile

GCT ATC TCC CCT GCC CTT GAG CAG CAG GGT ATC TTT GG 757
Ala Ile Ser Pro Ala Leu Glu Gln Gln Gly Ile Phe G1

gtatgtcaagcacgtcttacacattccgacatcgttgttgatgacgctctactgcacag T GGC GGA 823

GGT GCC GAT GGT
Gly Ala Asp Gly
AAC ATC GGT CTT
Asn Ile Gly Leu
AAC CTC TCC GTC
Asp Leu Ser Val
tcgtggccacag C

GCT
Ala
GAC
Asp
GCC
Ala
GCG
Ala
TTC
Phe
AAC
Asa
ATC
Ile
TCC
Ser
TTC
Phe
GTC
Val

CCC
Pro
GGT
Gly
GAC
Asp
CAC
His
GAC
Asp
GGC
Gly
GCC
Ala
GCG
Ala
CAG
Gln
GAC
Asp

CAG
Gln
CTT
Leu
GCA
Ala
ACC
Thr
TCG
Ser
ACG
Thr
GGC
Gly
TGC
Cys
TTC
Phe
TGC
Cys

gcaagcatag

GGT TTG ACC
Gly Leu Thr Val Asn Asp Ile Asp Gln Pro
gcagtactgatcaccggctatctcggacag TGC GTC GAG ACC CCC TTC CCC ACT CTC CCG 1775

e
CTC
Leu
GTC
Val
TCC
Ser
GTC
Val
ACT
Thr
ACC
Thr
GAG
Leu
GAG
Glu
GTG
Val
ACG
Thr
GTT
Val
GTC

TCC
Ser
GAC
Asp
GCT
Ala
ATT
Ile
GCC
Ala
CCC
Pro
CAG
Gln
GCT
Ala
CCA
Pro
TTC
Phe
TTC
Phe
TGG
Trp
TTC
Phe

y Gly Gly
ATC GCG ATC TTC TCA GAC ATC GAG ACC GCC TTC CAC CCC 877
Ile Ala Ile Phe Ser Asp Ile Glu Thr Ala Phe His Pro
GAG ATC GTC GAG CTG CAG AAG CCG TTC ATC GCG CGC CAC 931
Glu Ile Val Glu Leu Gln Lys Pro Phe Ile Ala Arg His
GAC TT gtgagcgttactgctattggtgtcaccataacaatatgctaac 994
Asp Ph
CAA TTC GCC GGA GCC ATC GGT GCC TCG AAC TGT GCG GGT 1049
Gln Phe Ala Gly Ala Ile Gly Ala Ser Asn Cys Ala Gly
GCC TTC GTC GGC CGC GTG GAT GCT ACT CAG CCC GCC CCC 1103
Ala Phe Val Gly Arg Val Asp Ala Thr Gln Pro Ala Pro
GAG CCG TTC CAC ACG CCC GAC CAG ATC TTC GCC CGC’CTC 1157
Glu Pro Phe His Thr Pro Asp Gln Ile Phe Ala Arg Leu
GGC GAG TTC GAC GAA ATC TTG ACC GTG TGG CTT CTC GTC 1211
Gly Glu Phe Asp Glu Ile Leu Thr Vla Trp Leu Leu Val
GCG GCC AAT GAC GTC GAC CCG ACT GTC CCC GGT TCG CCC 1265
Ala Ala Asn Asp Val Asp Pro Thr Val Pro Gly Ser Pro
GAG GTC TGG GAC ACG CAG TTC TTC GTC GAG GTC CTC CTG 1319
Glu Val Trp Asp Thr Gln Phe Phe Val Glu Val Leu Leu
CCC GGC ACC GGC GAC AAC CAG GGT GAG GTC GCG TCC CCG 1373
Pro Gly Thr Gly Asp Asn Gln Gly Glu Val Ala Ser Pro
CGC CTG CAG TCC GAC TTC GCG ATC GCG CGC GAC AGC CGC 1427
Arg Leu Gln Ser Asp Phe Ala Ile Ala Arg Asp Ser Arg
CAG TCG TTC GTC GAC AAC CAG CCC AAG GCG CAG GCC ATG 1481
Gln Ser Phe Val Asp Asn Gln Pro Lys Ala Gln Ala Met
CAC GAC CTT TCC ATC TTC GGC CAG GAC ATC AAC TCG CTC 1535
His Asp Leu Ser Ile Phe Gly Gln Asp Ile Asn Ser Leu

GAA GTT gtgcgtgtttctgctcctatagaacgttttagacaccaactaacca 1599

Glu
CCG
Pro
AAC

Val

ATT CCG GCA CCG CTC CAG GGC GTG ACC CAC TTC CCC GCT 1654
Ile Pro Ala Pro Leu Gln Gly Val Thr His Phe Pro Ala
GAC ATC GAC CAG CCT gtacgtatttctgatgcagccctagcctgtt 1715

Cys Val Glu Thr Pro Phe Pro Thr Leu Pro

ACT GAC CCC GGT CCC GCG ACC TCT GTC GCC CCC GT gtaagttttcagctatgatcgat 1833
Thr Asp Pro Gly Pro Ala Thr Ser Val Ala Pro Va
atgactagtcacttactacagccttttgtttcatag C CCC CTT CCG TGA GAGAATATATTAGAG 1897

1 Pro Len Pro Stop

GCGGTAACGCAAAGCTCATTTGAGCGTTGAAGGCGCGTGATGTGGATTAAAGCTTATGACTTCGATCTGG 1967
TGGTTGTGTTTTGTTGTATGGGTACTACCGCAGCTGCGGGACGTTGTATAACGGGTATACATGATGTTGA 2037
TGATC

2042

79

FIG. 5. Comparison of intron locations between the T. versicolor genes VLGZ
and VLGZ, and the P. chrysosporiwn genes, LIP2, UPS (6), and LIP6 (39). Solid

bars represent exon locations, solid lines represent intron locations.

80

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l-
G)

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l"
D

E
'u

l:
1:

E
'u

 

CHAPTER III

Regulation of the Lignin Peroxidase Gene
Transcripts in the White Rot Fungus

Trametes versicolor Grown in Wood

81

82

ABSTRACT

Temporal expression of the mRNA transcript levels for the lignin peroxidase
genes VLGI-VLG6 of the white-rot fungus Trametes versicolor, grown in a defined basal
medium with either pine (gymnosperm) or p0plar (angiospenn) wood powder as the
growth substrate, was studied. Transcripts of the lignin peroxidase genes VLGZ and
VLGS were detected in both p0plar and pine wood grown cultures, though only VLGS
transcripts were abundant during the early days of culturing on pine wood. Exogenous
NH4, glucose, and Mn(II) were added to the poplar grown cultures to study the effect of
these nutrients on lip gene expression. Transcripts of VLG3 could only be detected in
poplar wood grown cultures when excess nitrogen was added, and not under any other
experimental conditions. Also transcripts of VLGZ and VLGS were found to be
differentially regulated by the addition of excess glucose (56 mM) or nitrogen (22 mM)
to poplar wood cultures such that VLGZ was repressed by the addition of excess nitrogen
while VLGS was repressed by excess glucose levels. In contrast to previous observations
in defined medium, Mn(II) (at 15.4 ppm) failed to repress VLGZ or VLGS transcripts in
wood cultures. The differential expression of VLGZ and VLGS transcripts by growth in
poplar vs. pine wood, indicates that these genes are regulated by different environmental

signals present in angiospenn and gymnosperm wood.

83

INTRODUCTION

Wood is composed of three major biopolymers: cellulose, hemicellulose, and
lignin. Lignin forms a matrix with hemicellulose surrounding the cellulose fibrils (22).
The lignin in wood protects the plant polysaccharides cellulose and hemicellulose from
attack by pathogens (7). The complex and random nature of the linkages between the
phenyl propanoid subunits of lignin make it recalcitrant to degradation by many of the
soil and plant microbes (23). However, white-rot fungi, such as Trametes versicolor and
Phanerochaete chrysosporium. are known to degrade all three wood biopolymers to 002
(7). White-rot fungi degrade softwoods (gymnospermS) that contain guiacyl lignin
slower than hardwoods (angiosperms), which contain guiacyl/syringyl lignin (12’, 16).

The white-rot fungi produce two sets of enzymes, lignin peroxidases (LIP) and
manganese peroxidases (MNP) which are believed to be important in the degradation of
the lignin polymer (14, 26). UPS and MNPs utilize H202 to perform one electron
oxidations of the aromatic carbon centers of the lignin polymer(15, 20). LIPs directly
oxidize the aromatic carbon rings, while MNPs oxidize Mn(II) to Mn(III), which then
performs the oxidation of lignin (29). UPS and MNPs are only expressed in nitrogen or
carbon limited defined media (10, 11). LIP activity in P. chrysosporium can be repressed
and MNP activity enhanced by high levels (100 ppm) of Mn(II) in nitrogen limiting
media (4). The cloning of LIP genes from P. chrysosporium (8, 27, 28, 30) has shown
that a multigene family exists for LIPs and that LIP mRNA transcripts appear
concurrently with lignin degradation (27). Furthermore, LIP genes are differentially
regulated such that under nitrogen limitation the LIP genes LIP2, LIP5 and LIP6 are

actively transcribed, while under carbon limitation only LIP2 is actively transcribed (17).

84

An LIP multigene family has now been cloned from the important lignin
degrading fungus T. versicolor (Coriolus versicolor). Six LIP genes (VLGI through
VLG6) have been identified (2). This white-rot fungus also degrades lignin, and
expresses LIP and MNP activity, in either nitrogen or carbon limiting media (9, 18, 19).
We have studied the regulation of transcript levels for the VLG genes in nitrogen limiting
media, and have found that the major transcript detected is that for VLGZ. Also, in this
strain of T. versicolor, Mn(II) levels regulate LIP activity such that greater than 13 parts
per billion of Mn(II) represses LIP activity, and at 2.6 parts per million LIP activity is
completely absent (3).

Wood generally contains low levels of fixed nitrogen. Since lignin surrounds the
wood polysaccharides, it is possible that, carbon availability to the fungus is also limited
by the presence of lignin (22). Therefore, nitrogen or carbon limitation in defined media
has been assumed to realistically simulate the environment the fungi encounter when
growing on wood. However, it is not known if the most highly expressed LII; genes in
defined low nitrogen or carbon media are expressed at all in wood grown cultures. Also,
the finding of LIP gene repression in both T. versicolor (3) and P. chrysosporium (4) by
levels of Mn(II) normally found in wood (200 ppm), raises questions about the Mn(II)
regulation of LIP genes during growth in wood. Furthermore, considering the
differences in lignin content and degradation rates of angiospenn vs. gymnosperm wood,
we investigated the differences in the transcription patterns of the VLG genes when T.
versicolor was grown on white pine (a gymnosperm) vs poplar (an angiosperm) wood as
the substrate. Also, we report on the effect of addition of excess nitrogen, carbon, or

Mn(II) to LIP gene expression in wood-grown cultures by T. versicolor.

85

MATERIALS AND METHODS

Growth of T. versicolor in wood. T. versicolor strain (ATCC 12679) was used for all
experiments, and was maintained at 4° C. on malt extract slants. Milled poplar wood
(Poplus eugini) and milled eastern white pine wood (Pinus strobus) were obtained from
Dr. Hans Grethlein (Michigan Biotechnology Institute, East Lansing, MI). 5.0 grams dry
weight of a given milled wood was weighed out, soaked for 16 hours in 1.0 liter of
double distilled H20 at 4° C, filtered to remove the water, and stored at -20° C. The
soaked wood was added to the basal medium described previously (3, 21) at 5 g (dry
weight) wood per liter of medium, and sterilized by autoclaving. Except where stated
otherwise, exogenous NH4, glucose, and MnSO4 were excluded completely. T.
versicolor inoculum was grown in defined low nitrogen (2.2 mM NH4, 56 mM glucose)
medium for seven days and homogenized as previously described (3), and was added to
the wood media at 10% of the final volume. The inoculated medium was mixed and 10
ml aliquots were transferred aseptically to sterile 125 ml Erlenmeyer flasks. For
experiments where additional nitrogen, carbon, or Mn(II) were added, nitrogen was
added as diammonium tartrate to 11 mM; carbon was added as glucose to 56 mM; and
Mn(II) was added as MnSO4 to 15.4 ppm.

RNA extraction, Northern blots, and enzyme assays. Ten flasks were harvested on
days 4, 7, 10, 13, 16, 19, 22, and 25 after inoculation of the wood media. The contents of
the ten flasks were pooled, the extracellular fluid was separated from mycelium by
vacuum filtration, and the mycelium was frozen at -70° C until RNA extraction. LIP and
MNP assays on the extracellular fluid were performed as described previously (25).
Total RNA was extracted as described by Baker et al., (1), and 10 pg per lane was loaded
(as measured by UV260 absorbance) on a 1.2% formaldehyde agarose gel for the
experiments presented in Fig. 1-4, and 6. While 5 pg of total RNA was loaded per lane

86

for experiments presented in Fig. 5. Northern blots were prepared on nylon (Micron
Separations Incorporated, Westborough, MA), UV-crosslinked at 600 J/m2 in a
Stratalinker 1800 (Stratagene, La Jolla, CA), and hybridized to random primer 32?-
dCI'P labeled probes. Blots were hybridized at 42° C in a 50% forrnamide and 200 mM
NaCl hybridization buffer (24). Blots were washed at high stringency (twice for 15 min
each at room temperature with 5X SSC, 0.5% sodium dodecyl sulfate; twice for 15
minutes each at 37° C with 1X SSC, 1% SDS; three times for 15 minutes each at 65° C
with 0.1X SSC, 1% SDS). Blots were stripped of probe as described previously (24).

87

RESULTS

No LIP or MNP activity has been measurable in the extracellular fluid from
wood-grown cultures, possibly due to the low fungal biomass in these cultures, or
because of strong adsorption of UPS and MNPs to the wood substrates. Northern
blotting analyses showed that the VLGZ transcript (z1.3 kb) reaches maximum
abundance on days 10 and 22 (Fig. 1); however, the variation in transcript abundance
seen on different days may be in part due to variations in the loading of total RNA as
well. Therefore, the same blot was stripped of the VLGZ probe and was hybridized with
a ribosomal gene probe from Neurospora crassa (13) as a control for differences in
loading of total RNA between lanes (Fig 4A). This is a better measure of the
concentration of total RNA in each sample than UV260 absorbance because ribosomal
RNA content within the cell is relatively constant, and therefore variations! between
samples would indicate differences in loading of total RNA. The results showed
variation between samples, especially under loading in lane 5.

The above blot was stripped and re-hybridized, along with a northern blot
prepared with identical levels of total RNA from cultures grown on pine wood, with
VLGZ in order to observe the transcription patterns in pine wood as compared to those in
poplar. The results showed that the transcripts of VLGZ are also most abundant on day
22 when the fungus is grown on pine wood instead of poplar (Fig. 2). Fig. 4B indicates
that there are once again variations in loading of the total RNA from different samples of
pine grown cultures. However, it should be noted that transcript levels for VLGZ and
VLGS peak on different days for the samples from pine wood grown cultures which
suggests that the observed patterns of transcript abundance are not due to the variations

in amount of RNA loaded between samples.

The pattern of appearance for VLGS transcripts is similar to that for VLGZ when

88

poplar wood is used as the growth substrate (Fig. 3A) with peak transcript levels
observed on days 10 and 22. On the other hand, the pattern of transcription of VLGS is
different from VLGZ when pine wood is used as the growth substrate. In pine wood,
VLG5 transcript levels are higher during early days of culturing with peak transcript
levels on days 10 and 19, with the transcript levels on day 10 being higher than those on
day 19. Therefore, in pine wood grown cultures, VLGS transcript levels are higher
during the early days of culturing than during later days, while VLGZ transcript levels are
higher during the later days of growth (around 22 days) in pine wood cultures. Also,
transcripts for VLGS are apparent in pine wood grown cultures during the early days of
growth at a time when transcripts from VLGZ cannot be detected.

In order to compare the transcript levels of different VLG genes, we loaded RNA
samples from pine and poplar grown cultures into side by side lanes and prepared a
northern blot which was then probed with all the VLG genes. The results indicate that
the transcripts for VLGS are expressed early (day 10) in pine grown cultures while
transcripts for VLGZ are more abundant on day 19 (Fig. 5A and 5B). In poplar grown
cultures, transcript levels for bOth VLGZ and VLGS were higher on day 22 than on day
10. Northern blots of the pine and poplar cultures were then hybridized with the other
VLG genes cloned from this strain of T. versicolor. No transcripts could be found for
VLG], VLG3, VLG4, or VLG6.

Differential regulation of several lignin peroxidase genes has been shown to occur
in P. chrysosporium when nitrogen vs. carbon limiting defined media were used, and
Mn(II) levels of 2.6 ppm or greater were shown to repress the lignin peroxidase activity
of T. versicolor in defined low nitrogen media. Therefore, we investigated if the
molecular regulation of the LIP genes of T. versicolor would be altered by addition of
exogenous nitrogen, carbon, nitrogen and carbon together, or Mn(II) to cultures grown
on poplar. The results showed that the addition of 22 mM NH4 results in lower VLGZ

transcript levels as compared to the addition of 56 mM glucose (Fig. 6A, lanes 1-6) with

 

 

89

few differences apparent between early and late days of culturing. However, no VLGZ
transcripts were detected when high levels of both nitrogen and carbon (Fig. 6A, lanes 7-
9) were added. Furthermore, in contrast to previous results obtained using defined low
nitrogen media, addition of 5 ppm Mn(II) (Fig. 6A, lanes 10-12) did not repress VLGZ
transcripts.

The concentration of VLGS transcripts is somewhat different from VLGZ
transcripts under nitrogen and carbon repression conditions. Addition of 56 mM glucose
completely represses the appearance of VLGS transcripts on the later days of culturing
(Fig. 6B lanes 4-6), while addition of nitrogen (Fig. 6B, lanes 1-3) did not repress
transcript levels as effectively. As seen for VLGZ transcript levels, addition of 5 ppm
Mn(II) did not repress VLG5 transcripts levels (Fig. 6B, lanes 10-12). Moreover VLGS
transcript levels were relatively constant throughout the culturing period when compared
to the levels of VLGZ transcripts.

The results show that the addition of high levels of nitrogen (22 mM) to poplar
wood grown cultures of T. versicolor results in strong expression of VLG3 transcripts
(Fig. 6C lanes 1-3), while transcripts of VLG3 were not observed in poplar or pine grown
cultures not supplemented with nitrogen. However, supplementation of the poplar
medium with glucose, glucose plus 22 mM NH4, or Mn (11) did not result in expression
of VLG3 transcripts.

90

DISCUSSION

This study of the transcriptional abundance for the lip genes (VLGI to VLG6 of T.
versicolor grown on wood has revealed a complex pattern of expression of various VLG
genes. Both VLGZ and VLGS transcripts are seen in cultures grown in pine and poplar
wood, no other VLG transcripts could be detected in these cultures.

Transcript levels for VLGZ and VLGS appear to be differentially regulated by
addition of excess nitrogen and carbon to poplar wood grown cultures. Addition of
excess glucose to poplar grown cultures causes suppression of VLGS transcripts after day
7 (Fig. 6B lanes 4-6). Yet, addition of excess nitrogen to p0plar grown cultures did not
appear to suppress VLGS transcripts as severely as glucose addition did (Fig. 6B lanes 1-
3). In contrast to this, addition of excess nitrogen to poplar wood grown cultures
appeared to suppress VLGZ transcripts more than that seen upon addition 6f excess
glucose (Fig. 6A lanes 1-6). This result is similar to the differential gene regulation
found for P. chrysosporium in defined medium. Under nitrogen limiting conditions the
LIP genes LIP2, LIP5, and LIP6 are actively transcribed, while under carbon limitation
only LIP2 is actively transcribed (17). Also, it was found that the major transcript in
nitrogen limiting defined medium for T. versicolor was VLGZ, while transcripts for
VLGS were less abundant, if not absent all together (3). It would appear, therefore, that
the most highly expressed T. versicolor LIP genes in defined low nitrogen media are also
expressed in wood grown cultures, and that the genes are differentially regulated under
nitrogen or carbon limiting conditions.

Transcripts of VLGS are present in pine wood grown cultures during the first
weeks of growth, (Fig. 3B and Fig. SB) when transcripts of VLGZ are much less

abundant, if not absent altogether. Transcripts of VLGZ appear to become much more

abundant in pine wood grown cultures between 19-25 days. In poplar wood grown

91

cultures both VLGZ and VLGS transcripts are present during the early and the later days
of culturing. Since lignin depolymerization/degradation has been shown to be the rate
limiting step in the degradation of gymnosperm vs. angiosperm wood (12, 16), and lignin
peroxidases have been implicated in catalyzing the initial depolymerization of the lignin
polymer (26), it is interesting to find differences in the VLG transcript levels when the
two types of wood are used as growth substrates.

The appearance of VLGS transcripts with high levels of added nitrogen only in
wood grown cultures indicates that this gene is differentially regulated by nitrogen and
carbon limitation in poplar wood grown cultures. This is somewhat analogous to
previous observations with the nitrogen deregulated mutant of P. chrysosporium. der8-5
(5, 6). This mutant is deregulated for nitrogen limitation such that the lignin peroxidases
are induced in both low and high nitrogen media. In low nitrogen media, der8-5
produces the normal wild-type set of lignin peroxidases but in high nitrogen media a new
lignin peroxidase (LN) appeared. LN was not produced by the wild type strain of P.
chrysosporium under low or high nitrogen conditions (5, 6).

The repression of LIP activity in both P. chrysosporium and T. versicolor by 40
ppm (4) and 2.6 ppm (3) Mn(II) concentrations, respectively, in defined media has been
firmly established. The results reported here indicate that either soluble Mn(II) becomes
bound by the wood in a form not available to the fungus, or that in wood grown cultures
the lignin peroxidase genes are not as susceptible to repression by moderate levels of
Mn(II).

The differential regulation of transcripts of VLGZ, VLGS, and VLGS suggests that
there are environmental signals inducing these genes which are separate from the global
signal of nutrient starvation. That nutrient starvation is a pre-requisite to lignin
peroxidase gene expression is supported by the complete suppression of LIP gene
expression in cultures containing excess nitrogen and carbon. The appearance of VLG3

transcripts only in wood grown cultures containing excess nitrogen indicates that specific

92

VLG genes respond differentially to different nutritional signals. The expression of
different lignin peroxidase genes under different nutritional and environmental
conditions may explain, at least in part, LIP gene multiplicity in T. versicolor (2) and P.
chrysosporium (8, 27, 28, 30). This is exemplified by the presence of VLG3 transcripts
only under high nitrogen conditions in poplar wood. Since VLGZ is partially repressed
by nitrogen sufficiency, the discovery of a lignin peroxidase gene (VLGS) which is
expressed in the presence of high nitrogen, and si_lem in low nitrogen is significant.
More significantly, some of the signals may be specific to the type of wood
(gymnosperm vs. angiospenn) as illustrated by the differential expression of various VLG

genes of T. versicolor grown on poplar vs pine wood.

93

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Johansson, T., and P. O. Nyman. 1987. A manganese(II)-dependent
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Chem. Scand. B41:762-765.

Jonsson, L., T. Johansson, K. Sjostrom, and P. 0. Nyman. 1987. Purification
of ligninase isozymes from the white-rot fungus Trametes versicolor. ACI‘ A
Chem. Scand. B41:766—769.

Kersten, P. J., M. Tien, B. Kalyanaraman, and T. K. Kirk. 1985. The
ligninase of Phanerochaete chrysosporium generates cation radicals from
methoxybenzenes. J. Biol. Chem. 260:2609-2612.

21.
22.

23.

24.
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27.

28.

29.

30.

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Kirk, T. K., S. Croan, and M. Tien. 1985. Production of multiple ligninases by
Phanerochaete chrysosporiwn: effect of selected growth conditions and use of a
mutant strain. Enzyme. Microb. Technol. 8:27-32.

Kirk, T. K., and R. L. Farrell. 1987. Enzymatic "combustion": the microbial
degradation of lignin. Ann. Rev. Microbiol. 41:465-505.

Reddy, C. A. 1984. Physiology and biochemistry of lignin degradation, p. 558-
571. In M. J. Klug, and C. A. Reddy (eds.), Current Perspectives in Microbial
Ecology, Society for Microbiology, Washington, D.C.

Sambrook, Fritsch, and Maniatis. 1989. Molecular Cloning, second edition ed.,
vol 1-3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Tien, M. 1987. Properties of ligninase from Phanerochaete chrysosporium and
their possible applications. CRC Crit. Rev. Microbiol. 15:141-168.

Tien, M., and T. K. Kirk. 1983. Lignin degrading enzyme from the
hymenomycete Phanerochaete chrysosporium Burds. Science 221:661-663.

Tien, M., and C.-P. D. Tu. 1987. Cloning and sequencing of a cDNA for a
ligninase from Phanerochaete chrysosporium. Nature 326:520-523.

Walther, I., M. Kalin, J. Reiser, F. Suter, B. Fritsche, M. Saloheimo, M.
Leisola, T. Teeri, J. K. C. Knowles, and A. Fiechter. 1988. Molecular analysis
of a Phanerochaete chrysosporium lignin peroxidase gene. Gene 70:127-137.

Wariishi, H., H. B. Dunford, I. D. MacDonald, and M. H. Gold. 1989.
Manganese peroxidase from the lignin-degrading basidiomycete Phanerochaete
chrysosporiwn. J. Biol. Chem. 264:3335-3340.

Zhang, Y. Z., C. A. Reddy, and A. Rasooly. 1991. Cloning of several
lignin peroxidase (LIP)-encodin g genes: Sequence analysis of the LIP6

gene from the white-rot basidiomycete, Phanerochaete chrysosporium.
Gene 97:191-198.

96

FIG. 1. Temporal expression of the LIP gene, VLGZ, of T. versicolor in poplar
wood grown cultures. Lanes 1-8 contain 10 pg total RNA extracted,
respectively, from cultures on days 4, 7, 10, 13, 16, 19, 22 and 25 after
inoculation.

97

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98

FIG. 2. Regulation of expression of the transcripts of the LIP gene VLGZ of T.
versicolor in A. Poplar wood medium versus B. Pine wood medium. In both A.
and B. lanes 1-8 contained, respectively, 10 pg of total RNA extracted from
cultures on days 4, 7, 10, 13, 16, 19, 22 and 25 after inoculation was loaded on a
1.2% formaldehyde agarose gel. Northern blots were prepared and hybridized to
32P-CI‘P labeled VLGZ (3), and subsequently washed at high stringency as
described in materials and methods.

99

A B
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.

100

FIG. 3. Temporal expression of the transcripts of the LIP gene, VLGS, of T.
versicolor in A. Poplar wood medium, and B. Pine wood medium. Blots identical
to those in Fig. 2 were hybridized to labeled VLGS gene (2) as described in Fig.
2.

101

1A B
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‘—

102

FIG. 4. Differences in total RNA loaded between samples of A. poplar wood
grown cultures, and B. pine wood grown cultures. These blots are identical to
those in Figs. 2-3 except that these were hybridized with the ribosomal gene of
Neurospora crassa (13). Hybridization and washing conditions were identical to
those in Figs. 2-3.

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103

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104

FIG. 5. Comparison of lignin peroxidase gene transcript levels in pine vs. poplar
wood. Panels A. and B. represent identical northern blots probed, respectively,
with labeled VLGZ and VLGS. In each panel, lanes 1 and 2 contained 5 pg of
total RNA extracted, respectively, from pine wood grown cultures of T.
versicolor on days 13 and 19 after inoculation. Lanes 3 and 4 contain 5 pg of
total RNA extracted, respectively, from poplar induced cultures on days 10 and
22 after inoculation.

105

1234 1234

 

106

FIG. 6. Regulation of expression of lignin peroxidase genes as determined by
northern blot analyses. Identical blots were probed with VLGZ,(A.) VLGS. (B.)
and VLG3 (C.) Different lanes were loaded with total RNA from poplar grown
cultures containing 22 mM NH4 (lanes 1-3); 56 mM glucose (lanes 4-6); 22 mM
NH4 and 56 mM glucose (lanes 7-9); or 5 ppm Mn(II) (lanes 10-12). Cultures
were harvested on day 7 (lanes 1, 4, 7 and 10); day 14 (lanes 2, 5, 8 and 11); and
day 21 (lanes 3, 6, 9 and 12) after inoculation. Each lane contained 10 pg of total
RNA.

107

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SUMMARY AND CONCLUSIONS

108

109

SUMMARY AND CONCLUSIONS

Six lignin peroxidase genes were isolated from the white-rot fungus T rametes
versicolor by screening a genomic EMBL3 lambda phage library with a lignin peroxidase
cDNA clone, CLGS (1, 2) from the related white-rot fungus Phanerochaete
cMsosporier. The genes of T. versicolor occurred in linked sets within the lambda
clones: VLG] on clone 1; VLGZ, VLG3, and VLG4 on clone 2;and VLGS and VLG6 on
clone 3. The transcription of the genes was unidirectional within each linked set.

Sequence analysis of two of the genomic clones VLG] and VLGZ, and the cDNA
clone corresponding to VLGZ, revealed substantial similarities to the structural
organization of the lignin peroxidase genes of P. chrysosporium. The coding region of
VLGZ is interrupted by six small introns ranging in size from 51 to 68 bp. The putative
intron locations for VLG] and VLGZ are substantially similar. The mature proteins for
bOth VLGl and VLGZ are preceded by 25 and 26 amino acid signal sequences,
respectively, which end with the putative proteolytic cleavage site Arg-Arg. The mature
proteins coded for by both VLGI and VLGZ are substantially similar, but not identical, to
the LIP proteins isolated from a different strain of T. versicolor induced by carbon
limitation conditions (3, 4). The mature protein encoded by both VLGl and VLGZ
contains 341 amino acids (Mr = 36,714). 90.0% of the codons for VLGI, and 83.0% of
the codons for VLGZ end in G or C. Glycosylation at the single Ill-glycosylation site in
VLGl and at either, or both, of the N-glycosylation sites in VLGZ would increase the
molecular mass of these proteins to approximately 43-45 kd as previously reported for
the lignin peroxidase enzymes isolated from T. versicolor.

In comparison to the T. versicolor genes, the coding regions of the LIP genes of

110

P. chrysosporium are interrupted by eight to nine introns ranging in size from 50-62 bp.
The mature proteins are preceded by 27 to 28 amino acid signal sequences which end
with the amino acids Lys-Arg. The mature proteins are 344 amino acids long (Mr of
approximately 36,500 - 36,600). The codon usage for the LIP genes is also strongly
biased towards codons that end in G or C. Glycosylation at the N-glycosylation site(s)
would increase the size of the proteins to approximately 42 kd as has been measured for
the lignin peroxidase enzymes isolated from P. chrysosporium.

Substantial stretches of conserved amino acid sequences were found in the protein
encoded by VLGI versus those encoded by the lignin peroxidase genes of P.
chrysosporium and Phlebia radiata (1, 2, 5, 6). The regions between amino acids 62-81
and 188-208 correspond to the respective distal and proximal active histidine residues
already identified for peroxidases in general. However, Other long stretches of homology
at: 88-115, 162-174, 218-241, 273-286, and 335-354 have not had a function assigned to
them. These may correspond to substrate binding sites, or sequences conserved for
correct tertiary structure.

The coding sequence of VLGI is 79% homologous at the DNA level and 76%
homologous at the protein level to VLGZ. The VLGZ protein is only 58-63%
homologous at the amino acid level to the lignin peroxidases of P. chrysosporium and
62% homologous to the lignin peroxidase LGP3 of Phlebia radiata. VLGl is slightly
less homologous than VLGZ to the LIPS of P. chrysosporium, with 57-61% homology at
the amino acid level. But both are more homologous to P. chrysosporium gene LIP2
compared to the other LIP proteins of this organism.

The homology relationships between the lignin peroxidases of T. versicolor and
P. chrysosporium suggest the two multigene families arose independently by gene
duplication events from a common ancestral LIP gene. However the lip genes of P.
chrysosporium are more homologous to each other (70-80% homology at the amino acid

level) than they are to either VLGI or VLGZ (61% and 63% respectively). This is further

 

lll ‘

substantiated by DNA-DNA hybridization studies between LIP2 and LIP6, and VLGI-
VLG6 (see Appendix) which indicates that there are no separate homologs to either LIP2
or LIP6 amongst the VLG genes.

Two schools of thought currently exist as to the need for a multigene family in
the white-rot fungus P. chrysosporium. As already described, the first theory is that each
gene has a promoter controlled by a separate regulatory signal, such as nitrogen, carbon,
or Mn(II), and that each regulatory system responds to different environmental signals.
The second theory holds that each lignin peroxidase gene performs a different catalytic
activity or has a different substrate specificity, and that differential gene expression
allows only those LIP genes to be expressed as are needed for a particular lignin
substrate. According to this a different set of LIP genes are expressed when T. versicolor
(or P. chrysosporium or some other white-rot fungus) if grown on pine vs. poplar vs.
maple vs. some other wood species. Alternatively, it is possible that both the theories
proposed above may be true with regard to LIP gene regulation in white-rot fungi.

The discovery of two independently derived lignin peroxidase multigene families
in the white-rot fungi stimulated the question as to why so many lignin peroxidase genes
are necessary, and why this is a common feature in two genera of white-rot fungi? A
study of the differential gene regulation of the VLG genes was initiated so as to find out
which VLG genes were transcriptionally induced, and what the environmental signals for
that induction were. It was found that nitrogen limiting defined media triggered the
production of lignin peroxidase activity in this, strain of T. versicolor. The appearance
and levels of lignin peroxidase transcripts coincided with the appearance and levels of
lignin peroxidase in the culture supernatant and the transcripts appeared immediately
upon or following nitrogen limitation. VLGZ transcript was the major one present under
these conditions. Also Mn(II) concentrations above 13 parts per billion were found to
repress lignin peroxidase activity. Since wood is reported to have manganese levels of 5-

200 parts per million, this result called into question the relevance of either; a) lignin

112

peroxidase activity to lignin degradation, or b) these induction conditions to those
encountered during wood degradation in nature.

Study of the lignin peroxidase genes expressed by T. versicolor cultures grown in
defined media with poplar or pine wood added as the sole source of nitrogen and carbon
provided further information on the environmental signals regulating transcript levels in
this fungus. Transcript levels appeared to be higher (i.e. they were easier to detect and
were produced more reliably) when poplar or pine wood was used as the growth
substrate than when nitrogen limiting media were used.

VLGZ transcripts appear to be produced in an undulent temporal pattern during
growth on poplar wood; however, the possibility that this is due to variations in loading
of total RNA cannot be totally excluded. The reason for the observed temporal
variations is unknown, but could be related to a continuum of fungal transitions between
primary and secondary metabolism when growing on woody substrates. VLGZ transcript
levels appear to be greater on the later peak days as compared to transcript levels on
earlier days. The second major transcript in poplar wood grown cultures was VLGS,
which appeared to follow the same pattern of undulating transcript levels. The observed
inhibition of VLGZ and VLGS expression in defined media even by low levels of Mn(II)
was relieved by growth on pOplar wood Since the poplar wood already contained bound
manganese, and the soluble exogenous Mn(II) was added to replace any Mn(II) that was
lost by leaching during preparation of the wood samples, it can be concluded that the
lignin peroxidases of T. versicolor are expressed in wood which contains Mn(II).

VLGZ and VLGS were differentially regulated by growth in pine wood as opposed
to poplar wood. VLGZ transcripts could not be detected during the early days of growth
when a substantial increase in biomass was visually apparent. VLGS was the only
transcript detectable up until day 19 and appeared to have a undulent induction pattern
similar to both VLGZ and VLGS induced by poplar wood. However peak days were

separated by about nine days and VLGS transcript levels appeared to be decreasing as the

113

culture age increased.

Addition of excess nitrogen to poplar grown cultures resulted in lower levels of
VLGZ transcripts, but also allowed the expression of VLG3 transcripts; however, this did
not affect VLGS transcript levels. Furthermore, addition of excess glucose to poplar
wood grown cultures led to a moderate decrease in VLGS transcript levels while VLGZ
transcript levels were not lowered. Transcripts from VLG3 could not be detected in
poplar wood grown cultures with excess glucose added. These results suggest a possible
explanation for the existence of a lignin peroxidase multigene family in T. versicolor. It
has been observed that both VLGZ and VLGS transcripts are abundant in poplar wood
grown cultures. However, only transcripts from VLGS are abundant in early age pine
wood grown cultures. This indicates that p0plar wood contains different induction
signals than pine wood. Since pine contains a different type of lignin than poplar, the
intriguing possibility is that VLGS has a catalytic activity, or substrate specificity that is
more suited to gymnosperm lignin than is VLGZ. This may be verified by purification of
the VLGZ and VLG5 proteins and a comparative study made of their respective km and
Vmax values on pine and poplar lignin or model lignin compounds. If the above
hypothesis is correct, then VLGS would have different km or Vrnax values from VLGZ
towards lignin model compounds which more closely resemble gymnosperm lignin than
angiosperm lignin.

The need to induce lignin peroxidase activity under a wide variety of
environmental conditions may also be one of the causes for VLG gene multiplicity. This
is suggested by the presence of VLGS transcripts only under high nitrogen conditions in
p0plar wood. Since VLGZ expression is partially inhibited in the presence of excess
nitrogen, the discovery of a lignin peroxidase gene which is m by high nitrogen,
and sflm in low nitrogen is significant. The fact that VLGZ and VLG3 are closely
related, while VLGZ and VLGS are more distantly related tends to support the above

hypothesis.

114

In summary it can be concluded that transcript levels for VLGZ, VLG3, and VLGS
are differentially regulated by glucose and nitrogen levels present in poplar wood grown
cultures, and by unknown factors present in pine and poplar wood. It may be possible to
extract these unknown factors from pine and poplar wood, and add them to defined
media. This may lead to high level, reliable induction of transcripts from VLGZ, VLG3,
and VLGS, and possibly to higher level induction of these VLG enzymes. The
purification of the VLGZ and VLG5 lignin peroxidases would allow researchers to
determine if there are catalytic or substrate affinity differences between these two very
interesting enzymes. Since there are no homologs for either VLGZ, or VLGS amongst the
LIP genes of P. chrysosporium these results cannot be extended to the unanswered
questions surrounding the differential gene regulation observed in P. chrysosporiwn. It
would be interesting to determine if similar results could be obtained with the well

studied LIP genes of P. chrysosporium.

115

LIST OF REFERENCES

de Boer, H. A., Y. Z. Zhang, C. Collins, and C. A. Reddy. 1988. Corrigendum:
Analysis of nucleotide sequences of two ligninase cDNAs from a white-rot
filamentous fungusfhanerochaete chrysosporium. Gene 69:369.

de Boer, H. A., Y. Z. Zhang, C. Collins, and C. A. Reddy. 1987. Analysis of
nucleotide sequences of two ligninase cDNAs from a white-rot filamentous
fungus, Phanerochaete chrysosporium. Gene 60293-102.

Jonsson, L., T. Johansson, K. Sjostrom, and P. O. Nyman. 1987. Purification
of ligninase isozymes from the white-rot fungus Trametes versicolor. ACT A
Chem. Scand. B41:766-769.

Jonsson, L., O. Karlsson, K. Lundquist, and P. O. Nyman. 1989. Trametes
versicolor ligninase: isozyme sequence homology and substrate specificity. FEBS
Lett. 247:143-146.

Saloheimo, M., V. Barajas,‘ M-J. Niku-Paavola, J.K.C. Knowles 1989. A
lignin peroxidase-encoding cDNA from the white-rot fungus Phlebia radiata:
characterization in Trichoderma reesei. Gene 85 : 343-351.

Tien, M., and C.-P. D. Tu. 1987. Cloning and sequencing of a cDNA for a
ligninase from Phanerochaete chrysosporium. Nature 326:520-523.

 

APPENDIX

117

APPENDIX I

Determination of the Homology Relationships Within the Lignin Peroxidase Multigene
Family of Trametes versicolor, and Between the Lignin Peroxidase Subfamilies of
Trametes versicolor and Phanerochaete chrysospori um

The lignin peroxidase genes of Trametes versicolor VLGI-VLG6 can be grouped
into two subfamilies based upon DNA-DNA hybridization studies (Fig. l. A-C). Lignin
peroxidase genes VLG], VLGZ, VLG3, and VLG4 constitute one subfamily, while VLGS
and VLG6 make up a second subfamily. Interestingly VLGZ, VLG3 and VLG4 are linked
together within the genome, separated by 1.0-2.0 kb of non-LIP DNA. VLGS and VLG6
are likewise closely linked together within the genome.

P. chrysosporium is also known to have two subfamilies of lignin peroxidase
genes, represented by the LIP genes LIP2, LIP5, and LIP6. The existence of lignin
peroxidase multigene families in both P. chrysosporium and T. versicolor raises the
question if some of the genes between the two species might be homologs or share some
other common evolutionary history. The existence of homologs between the subfamilies
would be indicated by one set of VLG genes being more closely related to one of the P.
chrysosporium subfamilies than to the others. Since LIP2 and LIP6 represent separate P.
chrysosporium subfamilies, they were used to screen the VLGI-VLG6 genes of T.
versicolor for any clones which hybridized more intensely to either LIP2 or LIP6. The
results in Fig. 2. indicate that while VLG] through VLG4 hybridize more intensely to
LIP2 than either VLGS or VLGé, VLGI through VLG4 also hybridize more intensely to
LIP6. This indicates that VLG], VLGZ, VLGS, and VLG4 are more closely related to the
UP genes of P. chrysosporium. In particular VLGS is most closely related to the LIP
genes LIP2, and LIP6. However, no LIP2 or LIP6 homologs in the T. versicolor VLG
multigene family could be found. Furthermore the DNA-DNA hybridization studies and

stringency conditions necessary to promote hybridization between the LIP genes and the

118

VLG genes indicates that all the VLG genes are more homologous to each other than any
is to a particular LIP gene in P. chrysosporium. The collective evidence suggests that the
multiple lignin peroxidase genes presently known for P. chrysosporium and T. versicolor
arose by independent gene duplication events. A common ancestral gene would appear
to be most homologous to the P. chrysosporium gene LIP2 and the T. versicolor gene

VLG3 .

119

FIG. 1. Homology Relationships amongst the Lignin Peroxidase genomic clones of T.
versicolor. Genomic lambda clones (lane 1, clone 2; lane 2, clone 3; and lane 3, clone 1)
were cut with various restriction enzymes; lane 1, EcoRI and HindIII; lane 2, PstI; and
lane 3, BamHI; and electrophoresed in a 0.7% agarose gel in order to separate the
restriction fragments containing VLG genes. The VLG genes contained in each lane,
listed from the top of the lane to the bottom are: lane 1, VLG4, VLGZ and VLG3; lane 2,
VLG6 and VLGS; and lane 3, v1.01. The resulting southern blot was hybridized to 32P-
dCTP labelled probes of A. the 2.1 kb BamHI fragment from lambda clone 2 containing
VLGZ, B. the 2.5 kb PstI fragment from lambda clone 3 containing VLGS and, C. the
2.3 kb HindIII-EcoRI fragment from lambda clone 2 containing VLG3.

23

1

120

23

1

23

 

121

FIG. 2. Homology relationships between the LIP subfamilies of P. chrysosporium and T.
versicolor. Genomic lambda clones (lane 1, clone 2; lane 2, clone 3; lane 3, clone 4; and
lane 4, clone 1) were cut with various restriction enzymes; lane 1, HindIII; lane 2, Pstl;
lane 3, EcoRI; and lane 4, BamI-II; and electrophoresed in a 0.7% agarose gel in order to
separate the restriction fragments containing VLG genes. The VLG genes contained in
each lane, listed from the top of the lane to the bottom are: lane 1, VLGZ; lane 2, VLG6
and VLGS; and lane 3, VLG4 and VLG3; and lane 4, VLG]. The resulting southern blot
was hybridized to 32P-dcrp labelled cDNA probes of CLG4 (A.) and CLGS (B.) which

encode, respectively, lignin peroxidases H2 and H10 isozymes of P. chrysosporium.

122